CN101356269A - Methods for expression and purification of recombinant human growth hormone - Google Patents

Methods for expression and purification of recombinant human growth hormone Download PDF

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CN101356269A
CN101356269A CNA2005800444637A CN200580044463A CN101356269A CN 101356269 A CN101356269 A CN 101356269A CN A2005800444637 A CNA2005800444637 A CN A2005800444637A CN 200580044463 A CN200580044463 A CN 200580044463A CN 101356269 A CN101356269 A CN 101356269A
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hgh
amino acid
naturally encoded
peg
encoded amino
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英·布彻勒
里奇·刘
迈克尔·昂
斯图亚特·布赛尔
尼克·努森
丘霍松
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Ambrx Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH] (Somatotropin)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormones [GH] (Somatotropin)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Abstract

The present invention relates generally to the production, purification, and isolation of human growth hormone (hGH). More particularly, the invention relates to the production, purification, and isolation of substantially purified hGH from recombinant host cells or culture medium including, for example, yeast, insect, mammalian and bacterial host cells. The process of the present invention is also useful for purification of hGH linked to polymers or other molecules.

Description

The method of expression and purification of recombinant human growth hormone
The related application of cross reference
Present patent application requires the 60/638th of submission on December 22nd, 2004, the 60/655th of No. 616 U.S. Provisional Patent Application cases, submission on February 23rd, 2005, the 60/680th of No. 744 U.S. Provisional Patent Application case, submission on May 13rd, 2005, the 60/680th of No. 977 U.S. Provisional Patent Application cases and submission on October 17th, 2005, the right of priority of No. 977 U.S. Provisional Patent Application cases, its specification sheets all is incorporated herein.
Technical field
The present invention relates generally to production, the purifying of human growth hormone (hGH) and separate.Or rather, the present invention relates to from recombinant host production, purifying and separate the hGH of purifying substantially.
Background technology
Tethelin (GH) supergene family (Bazan, F.Immunology Today 11:350-354 (1990); Mott, H.R. and Campbell, I.D.Current Opinion in Structural Biology 5:114-121 (1995); Silvennoinen, O. and Ihle, J.N. (1996) SIGNALING BY THE HEMATOPOIETIC CytokineReceptors) represent one group of protein with analog structure feature.Each member of described protein families comprises 4 helical bundles.Though still have more family members to await identification, some family members comprise following each member: tethelin, prolactin, the placenta lactogen, erythropoietin (EPO), thrombopoietin (TPO), IL-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subelement), IL-13, IL-15, oncostatin M, the cilium human BDNF, leukaemia inhibitory factor, interferon-alpha, interferon-, IFN-, omega interferon, the τ Interferon, rabbit, the ε Interferon, rabbit, granulocyte-group's stimulating factor (G-CSF), granulocyte-scavenger cell group stimulating factor (GM-CSF), scavenger cell group stimulating factor (M-CSF) and heart nutrient substance 1 (CT-1) (" GH supergene family ").The member of GH supergene family has similar secondary and tertiary structure, although it has limited amino acid or dna sequence dna identity usually.Shared constitutional features is identified the newcomer of gene family easily.
The human growth hormone participates in the major part of normal human subject g and D and regulates.Described naturally occurring strand pituitrin is made up of 191 amino-acid residues and is had the roughly molecular weight of 22kDa.HGH shows a large amount of biological effects, especially comprises the activation and the Insulin-Like effect of linear growth (physique formation), lactation, scavenger cell and causes diabetes effect (people such as Chawla R., Ann.Rev.Med.34:519-547 (1983); Isaksson, people such as O., Ann.Rev.Physiol., 47:483-499 (1985); Hughes, J. and Friesen, H.Ann.Rev.Physiol., 47:469-482 (1985)).The structure of hGH is well-known (Goeddel, people such as D., Nature 281:544-548 (1979)), and the three-dimensional structure of hGH solves (de Vos, people such as A., Science 255:306-312 (1992)) by X-ray crystallography.Protein has the fine and close ball-like structure that comprises 4 amphiphilic bundles, begins to be called A-D from N-terminal, and described helical bundle passes through engagement of loops.HGH also contains 4 cysteine residues, its participate in two kinds of intramolecular disulfide linkage: C53 with C165 paired and C182 and C189 paired.Not glycosylation and in intestinal bacteria (E.coli), expressed (Chang, people such as C., Gene 55:189-196 (1987)) of hormone with secreted form.
Discerned a large amount of naturally occurring mutant of hGH.Described mutant comprises hGH-V (Seeburg, DNA 1:239 (1982); Incorporate the 4th, 446,235,4,670,393 and 4,665 of this paper by reference into, No. 180 United States Patent (USP)s) and contain 20-kDa hGH (people such as Kostyo, the Biochem.Biophys.Acta 925:314 (1987) of disappearance of the residue 32-46 of hGH; Lewis, people such as U., J.Biol.Chem., 253:2679-2687 (1978)).In addition, reported by after transcribe, after translate, secretion, metabolic process and other physiological processes and many hGH variants (Baumann, G., Endocrine Reviews 12:424 (1991)) of producing.The biological effect of hGH derives from the interaction of itself and specific cell acceptor.Hormone is the member of family who comprises the homologous protein of placenta lactogen and prolactin.Yet among the family member, hGH is uncommon, because its show specific specificity widely and with somatogenic (Leung, people such as D., Nature 330:537-543 (1987) acceptor or prolactin (Boutin through the clone, J. wait the people, Cell 53:69-77 (1988)) receptors bind.Based on structure and biochemical research, proposed to be used for lactagogue in conjunction with territory and somatogenic functional diagram (Cunningham, B. and Wells, J., Proc.Natl.Acad.Sci.88:3407 (1991)) in conjunction with the territory.The hGH acceptor is the member of hematopoiesis/cytohormone/growth factor receptors family, described family comprises some other growth factor receptorses, such as interleukin (IL)-3, interleukin-4 and Interleukin-6 acceptor, granulocyte scavenger cell group-stimulating factor (GM-CSF) acceptor, erythropoietin (EPO) acceptor and G-CSF acceptor.Referring to, Bazan, Proc.Natl.Acad.Sci USA 87:6934-6938 (1990).The member of cytohormone receptor family is contained 4 conserved cysteine residue and just is positioned at the tryptophane-Serine-X-tryptophane-Serine primitive of striding the diaphragm area outside.Think that conservative sequence relates to protein-protein interaction.Referring to people such as (for example) Chiba, Biochim.Biophys.Res.Comm.184:485-490 (1992).Interaction between the cell foreign lands of hGH and its acceptor (hGHbp) is one of hormone-receptor interaction of knowing the most.High-resolution X-ray crystallization data (Cunningham, people such as B., Science, 254:821-825 (1991)) show that hGH has two receptor binding sites and uses the different position on the molecule to come in regular turn in conjunction with two kinds of acceptor molecules.Two receptor binding sites are called position I and position II.Position I comprises the C-terminal of spiral D and part and the A-B ring of spiral A, and position II is contained the part of amino terminal region and the spiral C of spiral A.GH and its acceptor combine with position I at first in conjunction with and take place in regular turn.Then, position II meshes the 2nd GH acceptor, causes receptor dimerization and the activation that causes signaling path in the cell of hormone generation cell response.G120R replace has been introduced hGH mutain mass-energy among the II of position in conjunction with single hGH acceptor, but can not make two kinds of receptor dimerizations.Mutein is estimated may be by occupying the receptor site but do not make signaling path activation in the cell and take on hGH antagonist (Fuh, people such as G., Science 256:1677-1680 (1992)) in vitro.
Reorganization hGH is as therapeutical agent and be approved for a large amount of indications of treatment.The hGH deficiency can cause (for example) nanism, and described nanism is successfully treated above 10 years by the outside dispensing of hormone.The form of hGH deficiency (GHD) comprises paediatrics GHD, Childhood outbreak GHD and the outbreak of the growing up GHD that grows up that grows up.Except that the hGH deficiency, also ratified hGH is used for the treatment of renal failure (in children), Turner's synodrome (Turner ' sSyndrome) and AIDS patient's emaciation.In the recent period, Food and Drug Administration (FDA) approved is used for the treatment of non-GH dependency short-and slight in figure with hGH.Also hGH is used for the treatment of at present the elderly's aging, weakness, short bowel syndrome and congestive heart failure are studied.The target group of hGH treatment comprises children with special property sent out (ISS) of short and small stature and the grownup with GHD sample symptom.At present, reorganization hGH sells as daily injectable product, and 5 kinds of main products: Humatrope are arranged in the market TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono).Yet, use tethelin to be as the significant challenge of therapeutical agent, protein in vivo have the short transformation period and therefore its must by the subcutaneous injection of every day throw with to reach maximum validity people such as (, J.Clin.Endocrinol.Metab.81:1806-1809 (1996)) MacGillivray.Sizable effort concentrates on by reducing production costs, make dispensing to be more amenable for use with the patient, improve effect and security overview and producing other character that competitive edge will be provided and improves on the mode of dispensing of hGH agonist and antagonist.For example, Genentech and Alkermes sold Nutropin Depot in the past TM, the storage prescription of a kind of hGH is used for the paediatrics growth hormone deficiency.Allow the more dispensing of small frequency (every 2-3 week once rather than once a day) though store, it is also relevant with the adverse side effect such as the pain at biological usability that reduces and place, injection site, and withdraws from the market in 2004.In the recent period, another product P egvisomant TM(Pfizer) also ratify by FDA.Pegvisomant TMBe the genetically engineered analogue of hGH, it plays a part to be instructed to be used for the treatment of the highly selective growth hormone receptor antagonist (people such as van der Lely, The Lancet 358:1754-1759 (2001)) of acromegaly.Although Pegvisomant TMIn some amino acid side chain residues with polyoxyethylene glycol (PEG) polymer-derived, but product still throw every day with once, show that medical character is for best.Except that Pegylation with store the prescription, other dispensing routes that comprise the inhalant dosage form of hGH and oral dosage form be in clinical before and the commitment of clinical development, and also do not obtain the approval of FDA.Therefore, need to show that tethelin is active and provide longer serum half-life and the therefore polypeptide of the treatment transformation period of the better treatment level of hGH and increase.
In the recent period, reported the brand new technical in the protein science, it is hopeful to overcome proteinic site specific modification relative restrictions many and such as hGH.Specifically, novel composition has been added to prokaryotic organism Da Intestinal Escherichia (Escherichia coli, E.coli) (for example, people such as L.Wang, (2001), Science292:498-500) with eukaryote yeast saccharomyces cerevisiae (Sacchromyces cerevisiae, S.cerevisiae) (for example, people such as J.Chin, Science301:964-7 (2003)) protein biosynthesizing device, it has made the amino acid of non-genetic coding can incorporate intravital protein into.Offer the polynucleotide that body contains coding hGH polypeptide of constructing of host cell, described hGH polypeptide comprises selects codon and quadrature tRNA synthetic enzyme and/or quadrature tRNA to be used for non-naturally encoded aminoacid replacement to the hGH polypeptide.Use described method, response amber codon (TAG) will have novel chemistry, physics or biological property, comprise that photoaffinity labeling and intramolecular photosensitization amino acid, photocrosslinking amino acid are (referring to (for example) Chin, people such as J.W. (2002) Proc.Natl.Acad.Sci.U.S.A.99:11020-11024; And Chin, people such as J.W. (2002) J.Am. Chem.Soc.124:9026-9027), the amino acid of keto amino acid, the amino acid that contains heavy atom and the amino acid whose a large amount of novelties of glycosylation is incorporated in the protein in intestinal bacteria and the yeast effectively and with high frequency high fidelity.Referring to (for example), people such as J.W.Chin, (2002), Journal of the American Chemical Society124:9026-9027; J.W.Chin ， ﹠amp; P.G.Schultz, (2002), ChemBioChem3 (11): 1135-1137; People such as J.W.Chin, (2002), PNAS United States of America99:11020-11024; With L.Wang ， ﹠amp; P.G.Schultz, (2002), Chem. Comm.,1:1-11.All reference are all to incorporate into by reference.Described research is verified, might be optionally and introduce as usual for all functional groups in the amino acid that is found in 20 kinds of common, genetic codings and be chemically inert and may be used for effectively and chemical functional group that optionally reaction forms stable covalent linkage.The ability that the amino acid of non-genetic coding is incorporated in the protein allows introducing that naturally occurring functional group can be provided, such as the ε-NH of Methionin 2, halfcystine the chemical functional group of valuable surrogate of imino-of sulfhedryl-SH, Histidine.Known some chemical functional group is an inert for the functional group in the amino acid that is found in 20 kinds of common, genetic codings, but can dried net profit rope ground and react effectively to form stable key.
The covalently bound of hydrophilic polymer poly-(ethylene glycol) of being abbreviated as PEG is to increase the many biologically active molecules that comprise protein, peptide, and exactly water-soluble, the biological usability of hydrophobic molecule, increase its serum half-life, increase its treatment transformation period, regulate its immunogenicity, the method for regulating its biological activity or prolonging its cycling time.PEG be used in the medicine, on the artificial implantation widely and wherein biocompatibility, to lack toxicity and lack immunogenicity be during important other are used.In order to make the character maximization of desired PEG, the total molecular weight and the hydration status that are connected in the PEG polymkeric substance of biologically active molecules must be enough high, to give connecting relevant favorable characteristics with the PEG polymkeric substance usually, such as the water-soluble and circulating half-life that increases, can influence the biological activity of parent molecule simultaneously sharply.Any molecular weight of PEG can use by actual requirement, its include, but is not limited to about 100 dalton (Dalton, Da) to 100,000Da or be more (including, but is not limited to, is 0.1-50kDa or 10-40kDa sometimes) on request.Also can use side chain PEG, it includes, but is not limited to have the PEG molecule of each chain of MW in 1-100kDa (including, but is not limited to 1-50kDa or 5-20kDa) scope.
Usually by the reactive chemical functional group such as Methionin, halfcystine and histidine residues, N-terminal and carbohydrate partly are connected in biologically active molecules to the PEG derivative.Protein and other molecules often have the reactive moieties that can be used for the limited quantity that polymkeric substance is connected.Usually, be adapted to pass through polymkeric substance most and connect the position of modifying and in receptors bind, play an important role, and be to keep the biological activity of molecule necessary.Thereby, the indiscriminate bioactive remarkable reduction or even the complete loss that usually can cause polymer-modified molecule that be connected of the described reactive moieties on polymer chain and the biologically active molecules.People such as R.Clark, (1996), J.Biol.Chem.,271:21969-21977.The binding substances that has the enough polymericular weights that give the desired advantage of target molecule for formation, art methods is usually directed to being connected at random of many polymeric arms and molecule, and then the biological activity that has increased parent molecule reduces or even the risk of complete loss.
The domination of proteinic structure is formed for making the reactive moieties of the locus that the PEG derivative is connected with protein.The protein that comprises enzyme is by having universal architecture H 2The various sequences of the a-amino acid of N--CHR--COOH are formed.The amino part of an amino acid whose α (H 2N--) be connected in contiguous amino acid whose carboxy moiety (--COOH) to form amido linkage, it can be expressed as--(NH--CHR--CO) n--, subscript " n " can equal hundreds of or thousands of.The fragment of being represented by R can contain the reactive moieties that is connected that is useful on protein biological activity and PEG derivative.
For example, under the situation of amino acid lysine, in the ε position and in alpha position, exist--NH 2Part.ε--NH 2Free responding under the condition of alkaline pH value.Make most of technology in the field of protein derivedization be directed to exploitation with PEG and be used for connecting lysine residue ε--the NH that is present in protein 2The PEG derivative of part." Polyethylene Glycol and Derivatives for Advanced PEGylation ", Nektar MolecularEngineering Catalog, 2003, the 1-17 pages or leaves.Yet described PEG derivative all has the common limitation, and promptly it can not optionally be installed among the many lysine residues that exist on the protein surface of being everlasting.Lysine residue in being present in zymophore is under the important situation to (for example) protein active, or under the situation that lysine residue works in the interaction of adjusting protein and other biological molecule, as under the situation of receptor binding site, described limitation can be significant limitations.
Second and the complicacy of equal importance that are used for the existing method of protein Pegylation are that the PEG derivative can experience and the non-side reaction that requires that is different from the residue of desired described residue.Histidine contains the reactive imino-part that is expressed as--N (H)--on the structure, and and ε--NH 2Many chemical reactivity materials of reaction also can react with--N (H)--.Similarly, the side chain of amino acid cysteine has on the structure and is expressed as-the free sulfhydryl base of SH.In some cases, be oriented to the ε of Methionin--NH 2The PEG derivative at group place also can react with halfcystine, Histidine or other residues.Described reaction can produce PEG derive bioactive molecules complexity, heterogeneous mixture and emitting has the active risk of destroying the bioactive molecules that is used as target.Wish exploitation PEG derivative, it allows the chemical functional group is introduced single position in the protein, make then one or more PEG polymkeric substance can with the definition clear-cut on the protein surface and the bioactive molecules selectivity coupling at predictable special position.
Except that lysine residue, at other amino acid side chains that will comprise halfcystine, Histidine and N-end as the exploitation of the activated PEG reagent of target and carry out sizable effort.Referring to (for example), be incorporated herein by reference the 6th, 610, No. 281 United States Patent (USP)s and " Polyethylene Glycol and Derivatives forAdvanced PEGylation ", Nektar Molecular Engineering Catalog, 2003, the 1-17 pages or leaves.Can use known other technologies in site-directed mutagenesis and the affiliated field, optionally introduce cysteine residues in the proteinic structure at the position, and the free sulfhydryl base section that obtains can with the PEG derivatives reaction that has mercaptan-reactive functional groups.Yet described method is complicated, because the introducing of free sulfhydryl base makes gained protein expression, folding and stable complicated.Therefore, wish to have and a kind of the chemical functional group is introduced mode in the bioactive molecules, its make one or more PEG polymkeric substance can with optionally coupling of protein, simultaneously can be with sulfhedryl and see usually other chemical functional groups in the protein compatible (just, not in non-desired side reaction with sulfhedryl and other chemical functional groups that see usually in the protein mesh).
As seen from the sampling in affiliated field, be developed and be used to connect proteinic side chain, especially connect on the Methionin amino acid side chain--NH 2On part and the cysteine side chain-SH many described derivative partly, verified is problematic in it synthesizes and uses.Some derivatives and protein form unsettled key, described key suffer hydrolysis and therefore decompose, degraded or otherwise in aqueous environment, such as being unsettled in blood flow.Some derivatives form more stable key, but suffer hydrolysis before key forms, the meaning be the reactive group on the PEG derivative may be before connecting protein inactivation.Some derivatives are deleterious a little and so less being applicable in vivo.To such an extent as in fact some derivatives reactions are not effective too slowly.Some derivatives cause the loss of protein active by being connected in the position that produces activity of proteins.Some derivatives are not specific to its position with connection, the circulation ratio that it also can cause required active loss and lack the result.For overcoming and using the poly-relevant challenge of (ethylene glycol) part modifying protein, developed more stable (for example, United States Patent (USP) the 6th, 602, No. 498, it is to be incorporated herein by reference) or with molecule and lip-deep thiol moiety selective reaction (for example, United States Patent (USP) the 6th, 610, No. 281, it is to be incorporated herein by reference) the PEG derivative.In affiliated field, need far and away, in physiological environment, optionally react to form the PEG derivative till stablizing chemical bond up to requiring for chemically inert.
Therefore, exist the unsatisfied needs of the hGH polypeptide that is the pure form substantially that is applicable to that the human treatment uses are provided at present.In addition, need to produce medical rank hGH polypeptide according to efficient and the method for the scale operation that production cost is worth arranged.
Summary of the invention
The present invention relates generally to from recombinant host cell or substratum production and purifying hGH polypeptide.Or rather, the present invention relates to from the recombinant host production that includes, but is not limited to prokaryotic hosts, host bacterium or escherichia coli host and the purifying hGH polypeptide of purifying substantially.
In one embodiment, the invention provides and be used to separate the method for the hGH polypeptide of purifying substantially, the step that described method comprises is: (a) anion-exchange chromatography; (b) hydrophobic interaction chromatography (HIC).The purifying of Pegylation hGH polypeptide comprises following other step: (c) make hGH polypeptide and PEG reaction to form the hGH-PEG binding substances; (d) separate described hGH-PEG binding substances by anion-exchange chromatography.
In another embodiment, the invention provides and be used to separate the method for the hGH polypeptide of purifying substantially, the step that described method comprises is: (a) anion-exchange chromatography; (b) hydroxyapatite chromatography method; (c) hydrophobic interaction chromatography (HIC).The purifying of Pegylation hGH polypeptide comprises following other step: (d) make hGH polypeptide and PEG reaction to form the hGH-PEG binding substances; (e) separate described hGH-PEG binding substances by anion-exchange chromatography.
In one embodiment, recombinant host is to be selected from the group that is made up of prokaryotic cell prokaryocyte and eukaryotic cell.In one embodiment, recombinant host may comprise produces any host cell such as the insoluble subcellular components of inclusion body comprise the hGH polypeptide, it comprises (for example), yeast cell, mammalian cell, insect cell and comprise (for example) colibacillary bacterial cell.
The hydrophobic interaction chromatograph material that is applicable to method of the present invention may include, but is not limited to the matrix that replaces through alkyl or aryl, such as the matrix that replaces through butyl, hexyl, octyl group or phenyl, it comprises agarose, Sepharose, sepharose, Mierocrystalline cellulose, silica, dextran, polystyrene, poly-(methacrylic ester) matrix and includes, but is not limited to the polyvinylamine resin or the hybrid resin of poly-(methacrylic ester) matrix of replacing through butyl or phenyl.In a specific embodiment, the hydrophobic interaction chromatograph material may comprise the phenyl sepharose gel resin.
In one embodiment, the hGH polypeptide by the isolating purifying substantially of method described herein may include, but is not limited to, ripe hGH, ripe hGH variant, hGH polypeptide, hGH polypeptide variants and with poly-(ethylene glycol) bonded hGH.
At least a biological activity that may show in another embodiment, ripe hGH by the hGH polypeptide of the isolating purifying substantially of method of the present invention.
In another embodiment, the hGH polypeptide by the isolating purifying substantially of method of the present invention may be mammiferous.In a specific embodiment, the hGH polypeptide by the isolating purifying substantially of method of the present invention may be human.
In another embodiment of the present invention, the hGH polypeptide that obtains from the HIC step is covalently to be connected with water-soluble polymers.In certain embodiments, water-soluble polymers is poly-(ethylene glycol).In another embodiment, the hGH polypeptide comprises one or more non-naturally encoded amino acid.
The purifying that comprises non-naturally encoded amino acid whose hGH polypeptide expression and its Pegylation form provides the hGH molecule that changes in the site specific mode to be used for the treatment of purposes.The Pegylation of hGH polypeptide at natural amino acids coding place may cause the hGH polypeptide at the Pegylation at non-desired position and/or may be the Pegylation of the non-desired polypeptide of pollutent.It is unnecessary that the method for utilizing non-naturally encoded amino acid to be used for the site specific Pegylation of hGH polypeptide makes described purification step.
In another embodiment, owing to be used for and non-natural amino acid bonded unique chemical reaction, comprise combining of one or more non-naturals amino acid whose hGH polypeptide that exists and another molecule that includes, but is not limited to PEG, the hGH polypeptide of purifying substantially is provided.Comprise one or more non-naturally encoded amino acid whose hGH polypeptide with such as the combining of another molecule of PEG, may carry out with other purification techniques of before or after integrating step, carrying out, so that pure substantially hGH polypeptide to be provided.
Description of drawings
Fig. 1 shows the feed flow rate of 8 liters of fermentations.
Fig. 2 shows the fermenting process of 5 liters of scales.
Fig. 3, plate A and B show that the SDS-PAGE by the hGH polypeptide of pericentral siphon release and homogenization preparation analyzes.
Fig. 4 shows the technical process that is used for 5 liters of fermentations.
Fig. 5 shows the chemical structure of straight chain, 30kDa PEG.
Embodiment
Definition
Should be appreciated that the present invention is not subject to herein concrete grammar, scheme, the clone described, construct body and reagent and thereby may change.Should be appreciated that also term used herein only is in order to describe specific embodiment, and does not plan to limit category of the present invention, category of the present invention will only limit by accessory claim.
Press in this paper and the accessory claim and use, unless context clearly indication in addition, singulative " " and " described " comprise a plurality of reference substances.Therefore, (for example) be with reference to one or more described protein and comprise known its Equivalent of those skilled in the art with reference to a kind of " hGH ", or the like.
Unless otherwise defined, all technology used herein and scientific terminology have with the present invention under the common identical implication of understanding of technician in field.Although can with similar in appearance to or any method, equipment and the material that are equivalent to herein method, equipment and the material described be used for practice of the present invention or test, description preferable methods, equipment and material now.
Open case of all that mention and patent are to be incorporated herein by reference herein, describe in (for example) open case to reach to describe and disclose, and may get in touch the purpose of constructing body and method of use with the invention of current description.The open case of discussing herein that is provided only is its disclosure before the applying date of present application for patent.This paper will be interpreted as admitting that the inventor is owing to previous invention or owing to any other reason is authorized in advance than described disclosure.
The 11/046th, No. 432 U.S. patent application case is all to incorporate this paper by reference into.The 11/046th, No. 432 U.S. patent application case describe hGH naturally occurring aminoacid sequence, incorporate into non-naturally encoded amino acid whose position select and be used to make, method, composition, technology and the strategy of amino acid that purifying, sign and use are non-naturally encoded, non-naturally encoded amino acid hGH polypeptide and modified non-naturally encoded amino acid hGH polypeptide.
Comprise the mixture of polymer of amino acid or various polymkeric substance and do not mean that the polymer of amino acid of specified length by term used herein " protein ".Therefore, no matter use recombinant technology, chemistry or enzyme catalysis to synthesize and produce still natural existence, (for example) term peptide, oligopeptides and polypeptide all are included in the proteinic definition.Term also comprises by glycosylation, acetylizing, phosphorylation, and is like that and modify or deutero-peptide, oligopeptides and polypeptide.By defining herein, term " protein " is particularly including variant.Term " polypeptide ", " peptide " and " protein " alternately use in this article to refer to the polymkeric substance of amino-acid residue.In other words, similarly be applicable to the description and the proteinic description of peptide at the description of polypeptide, and vice versa.Term is applicable to that naturally occurring aminoacid polymers and one of them or an above amino-acid residue are non-naturally encoded amino acid whose aminoacid polymers.By used herein, the amino acid chain with any length that comprises full length protein contained in term, and wherein amino-acid residue is to connect by the covalency peptide bond.
By used herein, " tethelin " or " GH " should comprise have the human growth hormone with and the GH analogue, GH is with the merit heterogeneous, the GH stand-in, the GH fragment, hybridization GH protein, fused protein, oligomer and polymer, homologue, glycosylation type variant, variant, at least a bioactive described polypeptide and protein in splice variant and the mutein, irrelevant and in addition and include, but is not limited in vitro with the biological activity of above-mentioned substance, in vivo, recombinate (no matter by cDNA by the micro-injection of nucleic acid molecule, genome DNA, synthetic DNA still is that other forms of nucleic acid produces) method, synthetic method, its of transgenic method and gene activation method method synthetic or that make is irrelevant.Term " hGH polypeptide " or " hGH " are contained the hGH polypeptide that comprises one or more aminoacid replacement, interpolation or disappearance.Exemplary replacement on a plurality of amino acid positions in naturally occurring hGH comprises increasing the agonist activity, increases the proteolytic enzyme resistivity, polypeptide is changed into antagonist, regulate immunogenicity, regulate the replacement of receptors bind or the like,, all contain by term " hGH polypeptide ".
Concerning the naturally occurring GH aminoacid sequence and sophisticated naturally occurring GH aminoacid sequence and naturally occurring mutant of complete total length, respectively referring to herein SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.In certain embodiments, hGH polypeptide of the present invention is equal to SEQ ID NO:1 substantially, or SEQ ID NO:2, or any other sequence of SEQ ID NO:3 or growth hormone polypeptides.Discerned a large amount of naturally occurring mutant of hGH.Described mutant comprises hGH-V (Seeburg, DNA 1:239 (1982); Incorporate the 4th, 446,235,4,670,393 and 4,665 of this paper by reference into, No. 180 United States Patent (USP)s) and contain 20-kDa hGH (people such as Kostyo, the Biochem.Biophys.Acta 925:314 (1987) of disappearance of the residue 32-46 of hGH; Lewis, people such as U., J.Biol.Chem., 253:2679-2687 (1978)).Placental growth hormone is described in Igout, people such as A., Nucleic AcidsRes.17 (10): in 3998 (1989).In addition, reported by after transcribe, after translate, secretion, metabolic process and other physiological processes and many hGH variants of producing, it comprises through enzymolysis cracked variant or 2 chain variants (Baumann, G, Endocrine Reviews 12:424 (1991)).The nucleic acid molecule of coding hGH mutant and sudden change hGH polypeptide is well-known and includes, but is not limited to be disclosed in the 5th, 534,617; 5,580,723; 5,688,666; 5,750,373; 5,834,250; 5,834,598; 5,849,535; 5,854,026; 5,962,411; 5,955,346; 6,013,478; 6,022,711; 6,136,563; 6,143,523; 6,428,954; 6,451,561; Described nucleic acid molecule in 6,780, No. 613 United States Patent (USP)s and the U.S. Patent Application Publication case 2003/0153003; Described patent is incorporated this paper by reference into.Term " ahGH " also can be in order to refer to the having recombinant human growth hormone that non-naturally encoded amino acid whose location replaces.
Unless otherwise mentioned (that is to say, when explanation relatively be based on SEQ ID NO:1,3 or during other hGH sequences), otherwise to all of the amino acid position among the hGH that describes herein with reference to the position that is based among the SEQ ID NO:2.Be understood by those skilled in the art that, corresponding to SEQ ID NO:1,2,3 or any other GH sequence in the position of amino acid position can in any other hGH molecule, easily be discerned such as hGH syzygy, variant, fragment etc.For example, such as the sequence alignment program of BLAST can in order to comparison and be identified in the protein corresponding to SEQ IDNO:1,2,3 or other GH sequences in the particular location of position.Describe herein about SEQ ID NO:1,2,3 or amino acid whose replacement, the disappearance of other GH sequences or add also that desire refers to replacement, disappearance or the interpolation on the correspondence position in known hGH syzygy, variant, fragment or the like in the field that describe or affiliated herein, and contain by the present invention clearly.
The commercial formulation of hGH is sold with following title: Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono).
Term " hGH polypeptide " also comprises pharmaceutically acceptable salt and the prodrug of naturally occurring hGH, prodrug with salt, polymorphic form, hydrate, solvate, bioactive fragment, biological activity variant and steric isomer, and the agonist of naturally occurring hGH, stand-in and antagonist variant and its polypeptide syzygy.Comprising other amino acid whose syzygys that are positioned at N-terminal, C-terminal or both places is all contained by term " hGH polypeptide ".Exemplary syzygy includes, but is not limited to; (for example) the methionyl tethelin that is connected with N-terminal of methionine(Met) by the hGH of recombinant expressed generation; the syzygy (including, but is not limited to polyhistidyl or the affine fixed base of former Decision of resisting strenuously) that is used for the purifying purpose, the syzygy that forms with the serum albumin binding peptide and with the syzygy that forms such as sero-abluminous serum protein.The 5th, 750, No. 373 United States Patent (USP)s incorporating this paper by reference into, description is used to select to have the novel protein in conjunction with character (such as tethelin) of change and the method for antibody fragment variant for its acceptor molecule separately.Merge in the C-terminal territory that described method comprises the gene III coat protein that makes proteinic gene that coding pays close attention to and filobactivirus M13.
Various reference disclose by polymkeric substance combination or glycosylation modified polypeptide.Term " hGH polypeptide " comprises the polypeptide that combines with polymkeric substance such as PEG and one or more other derivatives that may comprise halfcystine, Methionin or other residues.In addition, the hGH polypeptide may comprise connexon or polymkeric substance, wherein the amino acid that combines with connexon or polymkeric substance may be alpha-non-natural amino acid according to the present invention, maybe may utilize such as with the affiliated field of Methionin or halfcystine coupling in known technology combine with natural amino acids coding.
The invention provides have multiple functional group, the hGH polypeptide of substituting group or part with include, but is not limited to the binding substances of other materials of following material: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; The photocrosslinking agent; Radionuclide; Cytotoxin compounds; Medicine; Affinity labelling; Photoaffinity labeling; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotides; Carbohydrate; Water-soluble branch-shape polymer; Cyclodextrin; Inhibition Yeast Nucleic Acid; Biomaterial; Nanoparticle; Spin label; Fluorophore, containing metal part; Radioactive segment; Novel functional group; With other molecules covalently or non-covalently interactional group; The photic part of shrouding; But intramolecular photosensitization part; Vitamin H; The derivative of vitamin H; The vitamin H analogue; The part of incorporating heavy atom into; The group of cleavable chemically; But the group of photo-cleavage; The side chain of elongation; The sugar that carbon connects; Redox active agent; Amino thioic acid sulfoacid; Deleterious part; Isotope-labeled part; The biophysics probe; Phosphorescent group; Chemiluminescent group; The close group of electronics; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Any combination of quantum dot, nanometer transmitter substance, radioactive nuleus, radioactivity transmitter substance, neutron capture agent or above-mentioned substance, or any other required compound or material.
Reported the polymer conjugates of hGH polypeptide.Referring to, (for example) incorporates the 5th, 849,535,6,136,563 and 6,608 of this paper, No. 183 United States Patent (USP)s by reference into.The 4th, 904, No. 584 United States Patent (USP)s disclose the polypeptide of Pegylation Methionin disappearance, and wherein at least one lysine residue has lacked or by any other radical amino acid replacement.WO 99/67291 discloses and is used for protein and PEG bonded method, and wherein at least one amino-acid residue on the protein lacks and under the condition that is enough to reach with protein bound, protein contacted with PEG.WO 99/03887 discloses the Pegylation variant of the polypeptide that belongs to the tethelin superfamily, and wherein cysteine residues is replaced by the non-essential amino acid residue in the regulation zone that is arranged in polypeptide.WO 00/26354 discloses and produces the method with the glycosylated polypeptides variant that reduces allergenicity, and when comparing with corresponding parent polypeptide, it comprises at least one other glycosylation position.The 5th, 218, No. 092 United States Patent (USP) discloses the modification of granulocyte group's stimulating factor (G-CSF) and other polypeptide, when comparing with natural polypeptides with box lunch, introduces at least one other carbohydrate chain.
The hGH polypeptide that comprises one or more aminoacid replacement, interpolation or disappearance contained in term " hGH polypeptide ".HGH polypeptide of the present invention can comprise with the modification of one or more natural amino acids to be modified together with one or more alpha-non-natural amino acids.The locational exemplary replacement of multiple amino acids in naturally occurring hGH polypeptide has been described, it includes, but is not limited to, regulate one or more bioactive replacements of hGH polypeptide, increase the solubility of agonist activity, increase polypeptide such as (and being not limited to), polypeptide is changed into antagonist, reduce the replacement of proteolytic enzyme susceptibility or the like and contain by term " hGH polypeptide ".In certain embodiments, the hGH polypeptide comprises bioactive interpolation, replacement or the disappearance of regulating the hGH polypeptide in addition.For example, interpolation, replacement or disappearance can be regulated the avidity to the hGH polypeptide receptor, regulate (including, but is not limited to increase or reduction) receptor dimerization effect, stablize the receptor dimerization thing, regulate circulating half-life, the adjustment of treatment transformation period, the stability of regulating polypeptide is regulated cracking by proteolytic enzyme, regulate dosage, adjustment release or biological usability promote purifying, or improve or change concrete dispensing route.
Similarly, the hGH polypeptide can comprise protease cracking sequence, reactive group, antibody binding domain (including, but is not limited to FLAG or poly-His) or other sequences based on avidity (including, but is not limited to FLAG, poly-His, GST etc.) or the link molecule (including, but is not limited to vitamin H) of detection (including, but is not limited to GFP), purifying or other characteristics of improving polypeptide.The hGH polypeptide can comprise secretory signal sequence.The example of secretory signal sequence includes, but is not limited to, and prokaryotic secretion signal sequence, eucaryon secretory signal sequence, 5 ' optimizing are used for the eucaryon secretory signal sequence of bacterial expression, novel secretory signal sequence, pectate lyase secretory signal sequence, Omp A secretory signal sequence and phage secretory signal sequence.The example of secretory signal sequence includes, but is not limited to, STII (protokaryon), Fd GIII and M13 (phage), Bgl2 (yeast) and the signal sequence bla that is obtained by transposon.
Homodimer, heterodimer, homology polymer and the heteromultimers that is connected also contained in term " hGH polypeptide ", it includes, but is not limited to directly be connected in identical or different non-naturally encoded amino acid side chain by non-naturally encoded amino acid side chain, be connected in natural amino acids coding side chain, or the described polymkeric substance that connects by connexon indirectly.Directly the hGH dipolymer that connects by the Cys-Cys disulfide linkage is described in Lewis, people such as U.J., J.Biol.Chem.252:3697-3702 (1977); Brostedt, P. and Roos, P., Prep.Biochem.19:217-229 (1989)) in.Exemplary connexon includes, but is not limited to such as poly-(ethylene glycol) or poly-dextran or has the water-soluble polymers of the polypeptide of all lengths.
Term " hGH polypeptide " also comprises glycosylated hGH, such as (and being not limited to) at the glycosylated polypeptide of any amino acid position, the glycosylation form that connects or connect by O by N of polypeptide.The variant that contains single Nucleotide change also is considered to the biological activity variant of hGH polypeptide.In addition, also comprise splice variant.Term " hGH polypeptide " also comprises hGH polypeptide heterodimer, homodimer, heteromultimers or the homology polymer of following material: connect or be expressed as the other biological bioactive molecule of any of fused protein or more than one hGH polypeptide or any other polypeptide, protein, carbohydrate, polymkeric substance, small molecules, connexon, ligand or any kind by chemical mode, and contain (for example) special disappearance or other keep bioactive modified polypeptides analogue.
Be understood by those skilled in the art that, can in any other hGH molecule, be easily discerned such as hGH syzygy, variant, fragment etc. corresponding to the amino acid position of the position in the concrete hGH sequence.For example, can and be identified in the protein particular location in order to comparison such as the sequence alignment program of BLAST corresponding to the position in the concrete GH sequence.
By used herein, " natural hGH " is defined as the hGH that comprises naturally occurring hGH, its analogue and variant, and it is for suitably folding and only contain suitable disulfide linkage.HGH also contain participate in two intramolecular disulfide linkage 4 cysteine residues: C53 with C165 paired and C182 and C189 paired, or the homologue of the analogue of hGH and the described amino-acid residue in the variant.Natural hGH is bioactive.
" insoluble hGH " refers to sedimentary or accumulative hGH, in other words is the hGH that is produced by recombinant host cell or in addition relevant recombinant host cell, and can take to have the biologically inert conformation of the inappropriate or unfashioned disulfide linkage of possibility.Insoluble hGH can be included in inclusion body or the refrangible body, that is to say, and under phase microscope, may or may not be visible.Insoluble hGH can be by by the known any method of those skilled in the art, makes solubility hGH become insoluble and produces.
" inappropriate folding hGH " refers to the hGH that is in the low bioactive conformation with inappropriate or unfashioned disulfide linkage.Inappropriate folding hGH can (but need not) be insoluble.
By used herein, term " hGH variant " comprises ripe hGH and hGH variant polypeptides." hGH variant " can be by the disappearance or the interpolation at the one or more position of (for example) one or more amino acid in mature protein, make of disappearance or the interpolation of one or more amino acid at the N-of mature protein end and/or C-end, and/or the replacement of one or more amino acid at the one or more position of mature protein produces and comprises the disappearance or the interpolation at the one or more position of one or more amino acid in mature protein, make of disappearance or the interpolation of one or more amino acid, and/or one or more amino acid are in the replacement at the one or more position of mature protein at the N-of mature protein end and/or C-end.For example, the hGH variant can produce by adding or lacking 10 amino acid, 5 amino acid, 3 amino acid or 1 amino acid at least at least at least at least.The hGH variant is translated modification after also can comprising, includes, but is not limited to glycosylation, acetylizing, phosphorylation, and is like that.Term " hGH variant " is particularly including mutant, allelic variant, homologue and the syzygy of (but being not limited to) ripe hGH sequence.The hGH variant also includes, but is not limited to peptide mimics or " class peptide ".Referring to WO 91/04282.
Term " purifying substantially " refers to following hGH polypeptide, it may be for substantially or be substantially free of just like in its naturally occurring environment under the situation of the hGH polypeptide that reorganization produces, that is to say seen in n cell or host cell normally follow or with the component of protein interaction.May comprise for the hGH that does not contain cellular material substantially have less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than the protein formulation of the contaminating protein matter of about 1% (with dry weight basis).When hGH polypeptide or its variant are during by the host cell recombinant production, protein may be with about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1% or still less amount and existing of dry cell weight.When hGH polypeptide or its variant are during by the host cell recombinant production, protein may be with about 5g/L of dry cell weight, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L or about 1mg/L or amount still less and is present in the substratum.Therefore, as analyzing by including, but is not limited to SDS/PAGE, RP-HPLC, the proper method of SEC and capillary electrophoresis is measured, " purifying substantially " hGH polypeptide of producing by method of the present invention can have at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, purity level at least about 70%, especially, at least about 75%, 80%, 85% purity level, more particularly, purity level at least about 90%, the purity level at least about 95%, at least about 99% or bigger purity level.
" recombinant host cell " or " host cell " refers to the cell that comprises external polynucleotide, no matter the method that is used to insert how, for example directly absorbs, transduction, f-cooperates or affiliated field in the additive method of known generation recombinant host cell.External polynucleotide can be maintained for example non-binding carrier format of plasmid, perhaps can be incorporated in the host genome.
By used herein, term " substratum " comprises can supporting or containing and comprises bacterial host cell, eukaryotic host cell, mammalian host cell, yeast host cell, insect host cell, plant host cell, Chinese hamster ovary celI, prokaryotic host cell, intestinal bacteria or any host cell of false Single born of the same parents bacterium host cell (Pseudomonas host cell) and any substratum, solution, solid, semisolid or the rigid support thing of cell content.Therefore, term can be contained host cell and grow in wherein substratum, for example the hGH polypeptide has been secreted into substratum wherein, is included in the substratum before or after the propagation step.Term also can (such as) in the hGH polypeptide is cell, produce and host cell is dissolved or division with under the situation that discharges the hGH polypeptide, contain the damping fluid or the reagent that contain the host cell lysate.
About protein refolding, " reductive agent " is to be defined as to maintain sulfhedryl in reduced state and the original molecule or any compound or the material of intermolecular disulfide linkage as used herein.The reductive agent that is fit to includes, but is not limited to dithiothreitol (DTT) (DTT), 2 mercapto ethanol, dithioerythritol, halfcystine, cysteamine (2-aminoothyl mercaptan) and reductive gsh.It will be apparent to those skilled in the art that multiple reductive agent is applicable to method of the present invention.
About protein refolding, " oxygenant " is to be defined as any compound or the material that can remove de-electronation from oxidized compound as used herein.The oxygenant that is fit to comprises (but being not limited to), the dithiothreitol (DTT) of Sleep-promoting factor B, Gelucystine, cystamine, oxidation, the erythritol of oxidation (oxidized erythreitol) and oxygen.It will be apparent to those skilled in the art that multiple oxygenant is applicable to method of the present invention.
As used herein, " denaturing agent " is to be defined as to cause that proteinic reversible separates folding any compound or material.The intensity of denaturing agent will be determined by the character and the concentration of concrete denaturing agent.But the denaturing agent that is fit to can be the combination of chaotropic agent, washing composition, organic solvent water-miscible solvent, phosphatide or two or more described medicament.The chaotropic agent that is fit to includes, but is not limited to urea, guanidine and Sodium Thiocyanate 99.Effectively washing composition such as sodium lauryl sulphate or Soxylat A 25-7 (for example can include, but is not limited to, Tween or Triton washing composition) strong washing composition, Sarkosyl, gentle non-ionic detergent (for example, digitonin), such as N-＞2,3-(two oily oxygen bases (dioleyoxy))-propyl group-N, N, the gentle cationic detergent of N-trimethyl aluminium, gentle ionic detergent (for example, Sodium cholic acid or sodium deoxycholate) or include, but is not limited to sulfonic acid betaine (Zwittergent (ampholytic detergent)), the zwitterionic detergent of 3-(3-courage amidopropyl) dimethyl amido-1-propane vitriol (CHAPS) and 3-(3-courage amidopropyl) dimethyl amido-2-hydroxyl-1-propane sulphite (CHAPSO).Such as acetonitrile, low-carbon (LC) alkanol (especially such as the C of ethanol or Virahol 2-C 4Alkanol) or the lower alkanes glycol (especially such as the C of ethylene glycol 2-C 4The alkane glycol) but solvent of organic water miscibility can be used as denaturing agent.Be applicable to that phosphatide of the present invention can be such as the naturally occurring phosphatide of phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositols or such as the synthetic phospholipid derivative or the variant of two hexanoyl phosphatidylcholines or Diheptanoylphosphatidylcholine.
By used herein, " folding again " describes the polypeptide that will contain disulfide linkage with regard to disulfide linkage from inappropriate folding or separate folding state-transition to natural or suitably any process, reaction or the method for folding conformation.
By used herein, " folding altogether " refers specifically at least two polypeptide interact with each other of use and makes and separate folding or inappropriate folding polypeptide and be transformed into natural, suitably folding process again, reaction or the method for folding polypeptide.
" non-naturally encoded amino acid " refers to the amino acid that is not one of 20 kinds of common amino acids or pyrroles's Methionin or seleno-cysteine.Other terms that can use with the free burial ground for the destitute with term " non-naturally encoded amino acid " are " non-natural amino acid ", " artificial amino acid (unnatural amino acid) ", " amino acid that non-natural exists " and its various versions that are connected with hyphen that be connected with hyphen and non-.Term " non-naturally encoded amino acid " also includes, but is not limited to, modification by natural amino acids coding (including, but is not limited to 20 kinds of common amino acids or pyrroles's Methionin and seleno-cysteine) (for example, after translate modification) amino acid that produces, but not for by translating the natural amino acid of incorporating the polypeptide chain in the growth into of mixture.The amino acid whose example that described non-natural exists includes, but is not limited to N-acetylglucosamine base (acetylglucosaminyl)-L-Serine, N-acetylglucosamine base-L-Threonine and O-phosphorylated tyrosine.
By used herein, term " water-soluble polymers " refers to any polymkeric substance that dissolves in the water-based solvent.The bonding of water-soluble polymers and hGH polypeptide can produce following change: include, but is not limited to respect to non-modified forms, increase or adjusting serum half-life or increase or adjustment of treatment transformation period, regulate immunogenicity, the association feature of the physics that adjusting such as gathering and polymer form changes receptors bind and changes receptor dimerization effect or poly effect.Water-soluble polymers can have or can not have its oneself biological activity, and can hGH be connected to other materials that include, but is not limited to one or more hGH polypeptide or one or more bioactive moleculess as connexon.The polymkeric substance that is fit to includes, but is not limited to polyoxyethylene glycol, polyoxyethylene glycol propionic aldehyde, its single C 1-C 10Alkoxyl group or aryloxy derivative (are described in and incorporate the 5th of this paper by reference into, 252, in No. 714 United States Patent (USP)s), mono methoxy-polyoxyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acid, the divinyl ether MALEIC ANHYDRIDE, N-(2-hydroxypropyl)-Methacrylamide, dextran, the glucan derivative that comprises dextran sulfate, polypropylene glycol, polyoxytrimethylene/ethylene oxide copolymer, the polyoxyethylene polyvalent alcohol, heparin, heparin fragment, polysaccharide, oligosaccharides, glycan, include, but is not limited to the Mierocrystalline cellulose and the derivatived cellulose of methylcellulose gum and carboxymethyl cellulose, starch and starch derivative, polypeptide, polyalkylene glycol and its derivative, the multipolymer of polyalkylene glycol and its derivative, polyvinyl ethyl ether and alpha-beta-poly-[(2-hydroxyethyl)-DL-l-asparagine, like that or its mixture.The example of described water-soluble polymers includes, but is not limited to polyoxyethylene glycol and serum albumin.
" N-terminal modification group " refers to aminoterminal any molecule that can be connected in polypeptide.Similarly, " C-terminal modification group " refers to any molecule of the C-terminal that can be connected in polypeptide.End modified group includes, but is not limited to various water-soluble polymerss, peptide or such as sero-abluminous protein, or increases other parts of the serum half-life of peptide.
By used herein, term " polyalkylene glycol " or " poly-(alkene glycol) " refer to polyoxyethylene glycol (poly-(ethylene glycol)), polypropylene glycol, polytetramethylene glycol and its derivative.Straight chain and branched chain polymer and the molecular-weight average between 0.1kDa and 100kDa contained in term " polyalkylene glycol ".Other one exemplary embodiment are listed in the goods catalogue of (for example) commercial supplier, in the catalogue that is listed in Shearwater Corporation " Polyethylene Glycol andDerivatives for Biomedical Applications " (2001).
Under term " functional group ", " active part ", " activating group ", " leavings group ", " reactive moieties ", " chemically reactive group " and " chemical reactivity part " are used for the field and refer in this article molecule uniqueness, definable part or unit.Described term in chemical field be a little synonym and be used for indicating herein carry out some function or active and can with the part of the molecule of other molecular reactions.
Term " key " or " connexon " refer to usually the group that forms owing to chemical reaction or key and covalent linkage normally in this article.The key of the hydrolysis-stable meaning is to be substantially stable in water and under effective pH value, to include, but is not limited under physiological condition, does not last time () the key or even indefinitely in an elongated segment time limit with the water reaction.Key meaning hydrolytically unstable or degradable is a degradable key in water or in the aqueous solution that comprises (for example) blood.Unsettled or the degradable key meaning of enzymatic is can be by the key of one or more enzyme liberating.By the understanding in the affiliated field, PEG and relevant polymkeric substance can comprise in the main polymer chain or the connexon group between the one or more functional end-group of main polymer chain and polymer molecule in degradable key.For example, by on biologically active agent, the reaction of PEG carboxylic acid or activated PEG carboxylic acid and alcohol radical and the ester bond that forms be hydrolysis release medicine under physiological condition usually.The degradable key of other hydrolysis includes, but is not limited to carbonic acid ester bond; The imine linkage that produces by the reaction of amine and aldehyde; React the phosphoric acid ester bond that forms by alcohol and phosphate; Hydrazone key for the reaction product of hydrazides and aldehyde; Be the acetal bonds of aldehyde with the reaction product of alcohol; Be the original acid ester key of manthanoate with the reaction product of alcohol; The peptide bond that forms at carboxyl by (including, but is not limited to) such as the amido of the end of the polymkeric substance of PEG and peptide; With the oligonucleotide key that forms at 5 ' hydroxyl of the phosphoramidite group of the end of polymkeric substance and oligonucleotide by (including, but is not limited to).
When term " bioactive molecules ", " biologically-active moiety " or " biologically active agent " when using in this article, the meaning is any physical properties or the biochemical property that can influence biosystem, path, molecule, or the influence interactional any material relevant with the organism that includes, but is not limited to virus, bacterium, phage, transposon, prion, insect, fungi, plant, animal and human's class.Specifically, by used herein, bioactive molecules includes, but is not limited to any material for the good order and condition of health that is used to diagnose, cure, relax, treat or prevent the disease of human or other animals or strengthen the mankind or animal in addition or spirit.The example of bioactive molecules includes, but is not limited to peptide, protein, enzyme, small-molecule drug, narcotic, soft medicine, carbohydrate, inorganic atoms or molecule, dyestuff, lipid, nucleosides, radionuclide, oligonucleotide, toxin, cell, virus, liposome, particulate and micella.Be fit to include, but is not limited to for the kind of the biologically active agent of the present invention's use, the toxin that medicine, prodrug, radionuclide, photographic developer, polymkeric substance, microbiotic, mycocide, antiviral agent, antiphlogistic drug, antineoplastic agent, cardiovascular agents, antianxiety agent, hormone, somatomedin, steroid medicament, microorganism obtain, like that.
" double functional copolymer " refers to the polymkeric substance that comprises two discrete functional groups, and described functional group can react to form covalent linkage or non covalent bond with other parts (including, but is not limited to amino acid side group) specifically.Have one can with the functional group of radical reaction on the concrete biologically active components, with another can with the difunctionality connexon of the group of radical reaction on second biological components, can comprise the binding substances of first biologically active components, difunctionality connexon and second biologically active components in order to formation.The many programs and the connexon that are used for all cpds is connected in peptide are known.Referring to (for example), No. the 188th, 256, European patent application; United States Patent (USP) the 4th, 671,958,4,659,839,4,414,148,4,699,784,4,680,338 and 4,569, No. 789, described patent is all incorporated this paper by reference into." polyfunctional poly compound " refers to the polymkeric substance that comprises two or more discrete functional groups, and described functional group can react to form covalent linkage or non covalent bond with other parts (including, but is not limited to amino acid side group) specifically.Double functional copolymer or polyfunctional poly compound can be any desired length or molecular weight, and can be selected so that concrete desired interval or the conformation between the one or more molecule that is connected in hGH to be provided.
At substituting group is during with its habitual chemical formula of from left to right writing explanation, and it similarly contains the chemically identical substituting group that obtains by writing structure from right to left, for example, and structure-CH 2O-is equivalent to structure-OCH 2-.
By used herein, term " serum half-life through the regulating " meaning is that the circulating half-life of modified hGH changes with respect to the positivity or the negativity of its non-modified forms.Serum half-life is to measure by various time point sample of blood, drawn behind throwing and hGH and the molecular conecentration of measuring in each sample.Serum-concentration mutual relationship in time can be calculated serum half-life.Wish that the serum half-life that increases has at least about twice, when still it can be facilitated gratifying dosage regimen or avoid toxic effect (for example), littler increase may be effective.In certain embodiments, increasing amount be at least about three times, at least about five times or at least about ten times.
By used herein, term " the treatment transformation period through the regulating " meaning is to change with respect to the positivity or the negativity of its non-modified forms the transformation period of the hGH polypeptide of treatment significant quantity.The treatment transformation period is by each time point after dispensing, measures the pharmacokinetics and/or the drug effect character of molecule and measures.The treatment transformation period that wish to increase can be facilitated concrete useful dosage regimen, concrete useful total dose or be avoided non-desired effect.In certain embodiments, the treatment transformation period that increases by the combining of the increase of the effectiveness that increases, decorating molecule and its target or reduction, pass through increase or reduction, or the increase of another parameter of non-decorating molecule or the mechanism of action or reduction and produce such as the molecular breakdown of the enzyme of proteolytic enzyme.
When being applied to nucleic acid or protein, term " separation " expression, nucleic acid or protein do not contain at least some other cellular components associating with it under state of nature, or nucleic acid or protein have been concentrated to the level of the concentration in producing greater than it in vivo or in vitro.It can be homogeneous state.Separated material can be drying regime or leather hard, perhaps is in solution state, includes, but is not limited to the aqueous solution.It can be the component of the medical composition of the pharmaceutically acceptable supporting agent that comprises other and/or vehicle.Purity and uniformity typically use such as the technique of analytical chemistry of polyacrylamide gel electrophoresis or high performance liquid chromatography and measure.For the protein that is present in the predominant in the preparation is purifying substantially.Specifically, separated gene separates from the proteinic open reading frame that side joint gene and coding are different from the gene of being paid close attention to.Term " purifying " expression, nucleic acid or protein produce band in fact in running gel.Exactly, it can mean, nucleic acid or protein be at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or bigger purity.
Term " nucleic acid " refers to deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and its polymkeric substance that is single stranded form or double chain form.Unless otherwise specifically limited, otherwise the nucleic acid of the analogue that contains known natural nucleotide contained in term, its have with reference nucleic acid similar combine character and to be similar to the mode metabolism of naturally occurring Nucleotide.Unless special in addition restriction, otherwise term also refers to the oligonucleotide analogs of the analogue (thiophosphatephosphorothioate, phosphoramidate, like that) that comprises PNA (peptide nucleic acid(PNA)), is used for the DNA of antisense technology.Unless otherwise instructed, otherwise the sequence that concrete nucleotide sequence is also implicitly contained its conservative variant of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and clearly indicated.Especially, degenerate codon replaces and can reach by producing following sequence, the 3rd position of one of them or more than one selected (or all) codon replaces (people such as Batzer, Nucleic Acid Res.19:5081 (1991) by mixing base and/or Hypoxanthine deoxyriboside residue; People such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); People such as Rossolini, Mol.Cell.Probes 8:91-98 (1994)).
Term " amino acid " refers to amino acid naturally occurring and that non-natural exists, and to be similar to amino acid analogue and the amino acid analog thing that naturally occurring amino acid whose mode works.Natural amino acids coding is 20 kinds of common amino acids (L-Ala, arginine, asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, L-iLeu, L-LEU, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan) and pyrroles's Methionin and seleno-cysteine.Amino acid analogue refers to the compound with basic chemical structure identical with naturally occurring amino acid, that is to say, has the α carbon compound that is incorporated into hydrogen, carboxyl, amino and R group, such as homoserine, nor-leucine, methionine sulfoxide, methionine(Met) methyl sulfonium.Described analogue has the R group (such as nor-leucine) of modification or the peptide main chain of modifying, but keeps the basic chemical structure identical with naturally occurring amino acid.
Amino acid can be mentioned by its common known 3 letter characters or by 1 letter character of being recommended by IUPAC-IUBBiochemical Nomenclature Commission in this article.Similarly, Nucleotide can be mentioned by its common received single alphanumeric codes.
" the conservative variant of modifying " is applicable to amino acid and nucleotide sequence.About concrete nucleotide sequence, " conservative modify variant " refers to the described nucleic acid of the identical or substantially the same aminoacid sequence of coding, or not during encoding amino acid sequence, refers to substantially the same sequence at nucleic acid.Since the degeneracy of genetic code, any given protein of many functional identical nucleic acid encodings.For example, codon GCA, GCC, GCG and GCU coded amino acid L-Ala all.Therefore, in each position that L-Ala is stipulated by codon, codon can be changed into described any corresponding codon and not change encoded polypeptide.It is " static variation " that described nucleic acid changes, and it is a kind of conservative changes in modification.Each nucleotide sequence of coded polypeptide is also described each possible static variation of nucleic acid herein.Those skilled in the art will realize that each codon in the nucleic acid (only removing usually the AUG for the codon of methionine(Met), with only outer for the TGG of the codon of tryptophane usually) can be modified to produce functional identical molecule.Therefore, the various static variation of nucleic acid encoding lies in described each sequence.
About aminoacid sequence, those skilled in the art will realize that, the single amino acid or amino acid whose discrete replacement, disappearance or the interpolation to nucleic acid, peptide, polypeptide or protein sequence of little per-cent that change, add or remove in the coded sequence are " the conservative variants of modifying ", wherein said change causes amino acid whose disappearance, amino acid whose interpolation or by aminoacid replacement amino acid like the chemofacies.Provide functional similar amino acid whose conservative getting to be represented as known to the those skilled in the art.The variant of described conservative modification except that polycrystalline variant of the present invention, plant between homologue and the allelotrope and do not get rid of polycrystalline variant of the present invention, plant between homologue and allelotrope.
Provide functional similar amino acid whose conservative getting to be represented as known to the those skilled in the art.Below eight kinds of groups contain the promising amino acid that is used for conservative replacement each other separately:
1) L-Ala (A), glycine (G);
2) aspartic acid (D), L-glutamic acid (E);
3) asparagine (N), glutamine (Q);
4) arginine (R), Methionin (K);
5) L-iLeu (I), L-LEU (L), methionine(Met) (M), Xie Ansuan (V);
6) phenylalanine (F), tyrosine (Y), tryptophane (W);
7) Serine (S), Threonine (T); With
8) halfcystine (C), methionine(Met) (M)
(referring to (for example), Creighton, Proteins:Structures and Molecular Properties (W HFreeman ﹠amp; Co.; Second edition (in December, 1993)
Under the situation of two or more nucleic acid or peptide sequence, term " identical " or per-cent " identity " refer to two or more identical sequences or subsequence.When comparing on comparison window or on the designated area and comparing maximum correspondence, by using following sequence comparison algorithm (or other algorithms of those skilled in the art's available) or measured by manual comparison and visual inspection, the per-cent that has identical amino-acid residue or Nucleotide as infructescence (that is to say, about 60% identity, about 65%, about 70%, about 75%, about 80%, about identity of 85%, about 90% or about 95% on the regulation zone), sequence is " identical substantially " so.Described definition also is meant the complementary sequence of cycle tests.Identity can be present on the zone of length at least about 50 amino acid or Nucleotide, or is present on the zone that length is 75-100 amino acid or Nucleotide, or when not stipulating, crosses over the full sequence of polynucleotide or polypeptide.
Concerning sequence compared, a common sequence was taken on and cycle tests reference sequences relatively.When using sequence comparison algorithm, with cycle tests and reference sequences input computer, (in case of necessity) specifies subsequence coordinate and specified sequence algorithm routine parameter.The program parameter of acquiescence can be used, maybe alternate parameter can be specified.Sequence comparison algorithm calculates the sequence identity per-cent of cycle tests with respect to reference sequences based on program parameter subsequently.
By used herein, " comparison window " comprises relating to having and is selected from by 20 to 600, common about 50 to about 200, more generally about 100 sections to the close position of any one quantity of about 150 groups that form, wherein after two sequences were by the best comparison, sequence can compare with the reference sequences of the close position with equal amts.The method of aligned sequences that is used for comparison is for known to the those skilled in the art.The optimal sequence comparison method that is used for comparison can (include, but is not limited to) the local homology's algorithm by Smith and Waterman (1970) Adv.Appl.Math.2:482c, homology alignment algorithm by Needleman and Wunsch (1970) J.Mol.Biol.48:443, the research of the searching similarity method by Pearson and Lipman (1988) Proc.Nat ' l.Acad.Sci.USA 85:2444, (Wisconsin Genetics Software Package is implemented in computerize by described algorithm, Genetics Computer Group, 575Science Dr., Madison, GAP among the WI, ESTFIT, FASTA and TFASTA), or by comparison and visual inspection (referring to people such as (for example) Ausubel, Current Protocols in Molecular Biology (1995 supplementary issue)) are carried out by hand.
An example that is suitable for measuring the algorithm of sequence identity and sequence similarity per-cent is BLAST and BLAST2.0 algorithm, its be described among people such as Altschul (1997) the Nuc.Acids Res.25:3389-3402 respectively and people (1990) J.Mol.Biol.215:403-410 such as Altschul in.Being used to carry out the software that BLAST analyzes is the open acquisition by NationalCenter for Biotechnology Information.The susceptibility and the speed of BLAST algorithm parameter W, T and X decision comparison.BLASTN program (being used for nucleotide sequence) is used 11 word length, 10 expected value (E), and M=5, the comparison of N=-4 and two chains is as default value.Concerning aminoacid sequence, the word length of BLASTP program use 3 and 10 expected value (E) are as acquiescence, and the BLOSUM62 matrix (referring to Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.USA 89:10915) of keeping the score uses 50 comparison value (B), 10 expected value (E), M=5, the comparison of N=-4 and two chains is as acquiescence.The BLAST algorithm is carried out when filter cuts out at " low-complexity " usually.
The BLAST algorithm is also carried out the statistical study (referring to (for example) Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5787) of two similaritys between the sequence.A kind of the measuring of the similarity that provides by the BLAST algorithm is minimum total probability (P (N)), and the coupling between two Nucleotide of its indication or the aminoacid sequence is with occurrent probability.For example, if the minimum total probability in the comparison of test nucleic acid and reference nucleic acid is less than about 0.2, less than about 0.01 or less than about 0.001, nucleic acid can be considered as similar to reference sequences so.
Phrase " optionally (or especially) hybridize to become " refers to when sequence is present in the complex mixture (including, but is not limited to general cell or storehouse DNA or RNA), under stringent hybridization condition, only with molecule in conjunction with, the duplex or hybridize in concrete nucleotide sequence.
Phrase " stringent hybridization condition " refers under as known low ionic strength in the affiliated field and hot conditions, the sequence of hybrid dna, RNA, PNA or other nucleic acid mimics or its combination.Usually, under stringent condition, probe will hybridize to its target subsequences in the complex mixture (including, but is not limited to general cell or storehouse DNA or RNA) of nucleic acid, but not hybridize to other sequences in the complex mixture.Stringent condition be sequence dependent and will be under different situations for different.Longer sequence is hybridized under higher temperature especially.The extensive guide of nucleic acid hybridization is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridizationwith Nucleic Probes is in " Overview of principles of hybridization and the strategy of nucleicacid assays " (1993).Usually, stringent condition is chosen as than under defined ionic strength pH value, the thermal melting point (T of particular sequence m) low about 5-10 ℃.T mBe 50% probe that adds to target and be in the hybridization of equilibrated target sequence (because target sequence is at T mDown excessive existence is so 50% probe is occupied under equilibrium state) temperature (under defined ionic strength, pH value and nucleic acid concentration).Stringent condition can be wherein pH 7.0 to 8.3 times, salt concn is less than about 1.0M sodium ion, usually about 0.01 to 1.0M Na ion concentration (or other salt) and concerning lacking probe (including, but is not limited to 10 to 50 Nucleotide), temperature is at least about 30 ℃ and concerning long probe (including, but is not limited to greater than 50 Nucleotide), and temperature is at least about 60 ℃ described stringent condition.Stringent condition also can be realized by the destabilizing agent that adds such as methane amide.Concerning selective cross or specific hybrid, positive signal can be at least 2 background hybridizations, optionally 10 background hybridizations.Exemplary stringent hybridization condition can be as follows: 50% methane amide, 5X SSC and 1%SDS, cultivate down at 42 ℃, or 5X SSC, 1%SDS, cultivate down, and under 65 ℃, in 0.2X SSC and 0.1%SDS, wash at 65 ℃.Described washing can be carried out more than 5,15,30,60,120 or 120 minutes.
By used herein, term " eukaryote " refers to and belongs to the organism that territory (phylogenetic domainEucarya) takes place in eucaryon kind system, such as animal (including, but is not limited to Mammals, insect, reptile, bird etc.), ciliate, plant (including, but is not limited to monocotyledons, dicotyledons, algae etc.), fungi, yeast, flagellate, microparticle insect order, protobiont etc.
By used herein, term " non-eukaryote " refers to non-most eukaryotes.For example, non-most eukaryotes can belong to Eubacteria (Eubacteria) and (include, but is not limited to colon bacillus (Escherichia coli), thermophilic bacterium (Thermus thermophilics), bacstearothermophilus (Bacillus stearothermophilus), pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), dislike breath utmost point hair bacillus (Pseudomonas putida) etc.) plant system generation territory, or primary class (Archaea) (includes, but is not limited to Methanococcus jannaschii (Methanococcus jannaschii), horizontal river virus (Methanobacteriumthermoautotrophicum), Halobacterium (Halobacterium) such as richly endowed salt bacterium (Haloferax volcanii) and salt bacillus specie (Halobacteriumspecies) NRC-1, the ancient green-ball bacterium (Archaeoglobus fulgidus) of glimmering, fireball bacterium (Pyrococcus furiosus), pick Yue Shi hot-bulb bacterium (Pyrococcus horikoshii), the quick hot bacterium of gas of thermophile bacteria (Aeuropyrum pernix) etc.) plant system the territory takes place.
By used herein, term " person under inspection " refers to animal, is Mammals in certain embodiments, and is human in other embodiments, and it is the target of treatment, observation or experiment.
By used herein, term " significant quantity " refer to that institute throws and the amount of non-natural amino acid polypeptides, it will alleviate one or more symptoms of disease, symptom or illness that desire treats to a certain extent.Can throw with the composition that contains herein the non-natural amino acid polypeptides of describing and be used for preventative, enhancing property and/or curative treatment.
Term " enhancing " meaning is increase or prolongs the effectiveness or the time length of desired effect.Therefore, about strengthening the effect of therapeutical agent, term " enhancing " refers to aspect effectiveness or time length, the ability of the effect of the other treatment agent in increase or the prolongation system.By used herein, " enhancing significant quantity " refers to the amount of the effect that is enough to strengthen another therapeutical agent in the desired system.When being used for the patient, the amount that is effective to described purposes will be looked the seriousness and the course of disease of disease, illness or symptom, above-mentioned therapy, patient's healthy state and the reaction of medicine and treatment doctor's judgement decided.
By used herein, term " modified " refers to any change that given polypeptide is carried out, such as change to the length of polypeptide, aminoacid sequence, chemical structure, the cotranslation of polypeptide modify or after translate modification.Term " (modified) " the form meaning is that the polypeptide of being discussed is optionally modified, and that is to say that in question polypeptide can be modified or do not modified.
Term " through after translate modification " refer to natural or non-natural amino acid after it incorporates polypeptide chain into, any modification that described amino acid takes place.Term contain in vivo the modifying of (only for example) cotranslation, cotranslation in vitro modification (such as, in the acellular system that translates), after translate in vivo modify and after the in vitro modification of translating.
In prophylactic application, the composition that will contain non-natural amino acid polypeptide throw with to the patient who emits the risk of suffering from concrete disease, illness or symptom concrete disease, illness or symptom sensitivity or opposite.Described amount is to be defined as " preventative significant quantity ".In described purposes, state of health, body weight that accurate amount is also looked the patient, like that and decide.The those skilled in the art is to determining that by normal experiment (for example, dosage escalation clinical trial) described preventative significant quantity has abundant understanding.
Term " through what protect " refers to " protecting group " or the existence of part that prevents that chemical reactivity functional group from reacting under some reaction conditions.Protecting group will be decided on the type of protected chemically reactive group.For example, if chemically reactive group is amine or hydrazides, protecting group can be selected from the group of tertiary butyl oxygen base carbonyl (t-Boc) and 9-fluorenes methoxycarbonyl (Fmoc) so.If chemically reactive group is a mercaptan, protecting group can be adjacent pyridyl disulfide so.If chemically reactive group is the carboxylic acid such as butyric acid or propionic acid, or hydroxyl, protecting group can be benzyl or such as the alkyl of methyl, ethyl or the tertiary butyl so.Under known other protecting groups in the field, comprise photolabile group, also the method and composition that can be used for describing or use herein herein with the method and composition of describing such as Nvoc and MeNvoc.Under known other protecting groups also can be used for describing herein in the field method and composition or use with the method and composition of describing herein.
Only for example, end-blocking/protecting group can be selected from:
Figure A20058004446300281
Allyl group Bn Cbz allyloxycarbonyl Me
Figure A20058004446300282
Et tertiary butyl TBDMS Teoc
Figure A20058004446300283
(C 6H 5) 3C——
Figure A20058004446300284
Figure A20058004446300285
Boc pMBn trityl ethanoyl Fmoc
Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, the third edition, John Wiley ﹠amp; Sons, New York, NY, in 1999, it is all to incorporate this paper by reference into.
In treatment is used, amount is enough to cure or stop at least in part the composition that contains (modified) non-natural amino acid polypeptide of the symptom of disease, illness or symptom, throw and the patient who has suffered disease, symptom or illness.Described amount is to be defined as " treatment significant quantity ", and will look the seriousness and the course of disease of disease, illness or symptom, above-mentioned therapy, patient's healthy state and the reaction of medicine and treatment doctor's judgement decided.The those skilled in the art is to determining that by normal experiment (for example, dosage escalation clinical trial) described treatment significant quantity has abundant understanding.
Term " treatment " is often referred to preventative and/or therapeutic treatment.
The non-naturally encoded amino acid polypeptide of Ti Chuing can comprise through isotope-labeled compound herein, and it has the atom of one or more atomic substitutions by having the atomic mass that is different from common atomic mass of occurring in nature or total mass number or total mass number.Can incorporate the isotropic substance that isotopic example in the The compounds of this invention comprises hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine into, such as being respectively, 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36Cl.Some of Miao Shuing be through isotope-labeled compound herein, for example such as 3H and 14The emitting isotope of C is incorporated described compound wherein into, applicable to medicine and/or matrix organization's distribution calibrating.In addition, use such as deuterium, just, 2The isotopic replacement of H can provide some by greater metabolic stability more and the treatment advantage that obtains, for example dosage requirement of in vivo transformation period of Zeng Jiaing or reduction.
Include, but is not limited to the part of the composition that all isomer of diastereomer, enantiomer and its mixture are regarded as describing herein.Other or in addition among the embodiment, non-naturally encoded amino acid polypeptide throw with the organism that needs to produce metabolite after metabolism, subsequently described metabolite is comprised the desired effect of desired treatment effect in order to production.In other or additional embodiments is the active metabolite of non-natural amino acids coding polypeptide.
In some cases, non-naturally encoded amino acid polypeptide can be the tautomeric forms existence.In addition, the non-naturally encoded amino acid polypeptide of the Miao Shuing form of solvation not herein, and with exist such as water, the formed solvation form of the suchlike pharmaceutically acceptable solvent of ethanol.The solvation form also is considered as being disclosed in herein.Those skilled in the art will realize that some compounds herein can some tautomeric forms exist.The part of the composition that all described tautomeric forms are regarded as describing herein.
Embodiment
I. explanation
The hGH molecule that comprises at least a alpha-non-natural amino acid is provided among the present invention.In certain embodiments of the present invention, the hGH polypeptide with at least a alpha-non-natural amino acid comprises and translates modification after at least a.In one embodiment, translate after at least a to modify and comprise the chemical process of utilizing known to the those skilled in the art that is suitable for concrete reactive group, the molecule that includes, but is not limited to following material that will comprise second reactive group is connected at least one alpha-non-natural amino acid that comprises first reactive group: mark, dyestuff, polymkeric substance, water-soluble polymers, the derivative of polyoxyethylene glycol, the photocrosslinking agent, radionuclide, cytotoxin compounds, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotides, carbohydrate, water-soluble branch-shape polymer, cyclodextrin, inhibition Yeast Nucleic Acid, biomaterial, nanoparticle, spin label, fluorophore, metallic part, radioactive segment, novel functional group, covalently or the group of non-covalent and other interactions of molecules, the photic part of shrouding, but the part that actinic radiation excites, the intramolecular photosensitization part, vitamin H, the derivative of vitamin H, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, the side chain of elongation, sugar through the carbon connection, the redoxomorphism promoting agent, amino thioic acid sulfoacid, deleterious part, the part of isotropic substance ground mark, the biophysics probe, phosphorescent group, chemiluminescent group, the close group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable mark, small molecules, quantum dot, the nanometer transmitter, the radioactive nuleus thuja acid, the radioactivity transmitter, neutron capture agent, or any combination of above-mentioned substance or any other required compound or material.In some embodiment of modified hGH polypeptide of the present invention, use comprises at least one alpha-non-natural amino acid (including, but is not limited to contain the alpha-non-natural amino acid of ketone) of translating modification after at least a, translates to modify after wherein at least a to comprise the carbohydrate part.In certain embodiments, after translate that to modify be at eukaryotic cell or in non-eukaryotic cell, in vivo carry out.
In certain embodiments, protein comprises at least a by a kind of host cell, translates modification after carrying out in vivo, and translating modification after wherein can not normally be undertaken by another kind of host cell type.In certain embodiments, protein comprises at least a by eukaryotic cell, translates modification after carrying out in vivo, and translating modification after wherein can not normally be undertaken by non-eukaryotic cell.After translate modification example include, but is not limited to glycosylation, acetylizing, acylation, lipid-modified, palmitoylation effect, cetylate interpolation, phosphorylation, the glycolipid key is modified, and is like that.In one embodiment, translate after to modify to comprise and by GlcNAc-asparagine key oligosaccharides is connected to asparagine (including, but is not limited to wherein, oligosaccharides comprises (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc, like that).In another embodiment, translate after to modify to comprise oligosaccharides (including, but is not limited to Gal-GalNAc, Gal-GlcNAc etc.) is connected to Serine or Threonine by GalNAc-Serine key, GalNAc-Threonine key, GlcNAc-Serine key or GlcNAc-Threonine key.In certain embodiments, protein of the present invention or polypeptide can comprise secretion or positioning sequence, Kang Yuan Decision decides disjunction mark label, FLAG label, polyhistidyl label, GST syzygy and/or like that.The example of secretory signal sequence includes, but is not limited to prokaryotic secretion signal sequence, eucaryon secretory signal sequence, 5 ' optimizing and is used for the eucaryon secretory signal sequence of bacterial expression, novel secretory signal sequence, pectate lyase secretory signal sequence, Omp A secretory signal sequence and phage secretory signal sequence.The signal sequence bla that the example of secretory signal sequence includes, but is not limited to STII (protokaryon), Fd GIII and M13 (phage), Bgl2 (yeast) and obtained by transposon.
Protein of being paid close attention to or polypeptide can contain at least a kind, at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds, or the alpha-non-natural amino acid more than 10 kinds or 10 kinds.Alpha-non-natural amino acid can be identical or different, for example, in protein, may exist comprise 1,2,3,4,5,6,7,8,9, the different sites more than 1,2,3,4,5,6,7,8,9,10 or 10 of the different alpha-non-natural amino acid more than 10 kind or 10 kinds.In certain embodiments, at least a (but less than all) the concrete amino acid that is present in the proteinic naturally occurring version replaces through alpha-non-natural amino acid.
The invention provides method and composition, provide specifically to comprise at least a non-naturally encoded amino acid whose hGH based on tethelin.At least a non-naturally encoded amino acid introduced make among the hGH and relate to (including, but is not limited to) with one or more non-naturally encoded amino acid whose particular chemical reactions but do not used simultaneously with the chemical action that combines that 20 seed amino acids of common existence react.In certain embodiments, comprise that non-naturally encoded amino acid whose hGH connects by non-naturally encoded amino acid whose side chain or binding in water-soluble polymers such as polyoxyethylene glycol (PEG).The invention provides the high efficiency method that is used for by PEG derivatives selectively modifying protein, it relates to response and selects the amino acid of codon with non-genetic coding, include, but is not limited to contain the functional group or the substituent described amino acid that include, but is not limited to the ketone part that in 20 kinds of natural amino acid of incorporating into, can not see, selectivity is incorporated in the protein and is come the described amino acid of follow-up modification by the reactive PEG derivative that is fit to.In case after incorporating into, subsequently just can be by utilizing known to the those skilled in the art, the concrete functional group or the substituent chemical process that are suitable for existing in the non-naturally encoded amino acid are come the modified amino acid side chain.Multiple known chemical process is applicable to that the present invention incorporates water-soluble polymers in the protein into.
The invention provides have multiple functional group, the hGH polypeptide of substituting group or part with include, but is not limited to the binding substances of other materials of following material: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; The photocrosslinking agent; Radionuclide; Cytotoxin compounds; Medicine; Affinity labelling; Photoaffinity labeling; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotides; Carbohydrate; Water-soluble branch-shape polymer; Cyclodextrin; Inhibition Yeast Nucleic Acid; Biomaterial; Nanoparticle; Spin label; Fluorophore, metallic part; Radioactive segment; Novel functional group; Covalently or non-covalentlyly with the group of other interactions of molecules; The photic part of shrouding; But the part that actinic radiation excites; The intramolecular photosensitization part; Vitamin H; The derivative of vitamin H; The vitamin H analogue; The part of incorporating heavy atom into; The group of cleavable chemically; But the group of photo-cleavage; The side chain of elongation; Sugar through the carbon connection; The redoxomorphism promoting agent; Amino thioic acid sulfoacid; Deleterious part; Isotope-labeled part; The biophysics probe; Phosphorescent group; Chemiluminescent groups; The close group of electronics; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable mark; Small molecules; Quantum dot; The nanometer transmitter; Radionuclide; The radioactivity transmitter; Neutron capture agent; Or any combination of above-mentioned substance, or any other required compound or material.
Under determine that PEG can be used for the surface of modified biological material (referring to No. the 6th, 610,281, (for example) United States Patent (USP) in the field fully; Mehvar, R., J.Pharm Pharm Sci., 3 (1): 125-136 (2000), it is to incorporate this paper by reference into).The PEG derivative can be by reactive partly direct and polymkeric substance binding.Perhaps, the PEG derivative can prepare by being connected to habitual activatory polymkeric substance at the linking agent that an end has a reactive part, so that the polymkeric substance of gained has reactive part at its end.Perhaps, have the water-soluble polymers of at least one active nucleophilic or electrophilic part and the reaction that has the linking agent of reactive group at an end, so that form covalent linkage between PEG polymkeric substance and linking agent, and reactive group is positioned at the end of polymkeric substance.Comprise amine, mercaptan, hydrazides, hydrazine, alcohol, carboxylicesters, aldehyde, ketone, thioesters, suchlike nucleophilic and electrophilic part are known to the those skilled in the art.The PEG derivative can be used for changing biological fitness, stability, solubility and the surface of shortage immunogenicity outbalance and the character of molecule, the PEG derivative is connected under the proteinic ratio mode of previously known has more optionally mode in the field and provide simultaneously.
II. tethelin supergene family
Following protein comprises by the described protein of the coded by said gene of tethelin (GH) supergene family (Bazan, F., Immunology Today 11:350-354 (1990); Bazan, J.F.Science 257:410-413 (1992); Mott, H.R. and Campbell, I.D., Current Opinion in Structural Biology 5:114-121 (1995); Silvennoinen, O. and Ihle, J.N., SIGNALLING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS (1996)): tethelin, prolactin, the placenta lactogen, erythropoietin (EPO), thrombopoietin (TPO), IL-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subelement), IL-13, IL-15, oncostatin M, cilium human BDNF (CNTF), leukemia suppresses factor (LIF), interferon-alpha, interferon-, the ε Interferon, rabbit, IFN-, omega interferon, the τ Interferon, rabbit, granulocyte-group's stimulating factor (G-CSF), granulocyte-scavenger cell group stimulating factor (GM-CSF), scavenger cell group stimulating factor (M-CSF) and heart nutrient substance-1 (CT-1) (" GH supergene family ").Other members that can expect described gene family will discerned by gene clone and sequencing in the future.Although the member of GH supergene family has limited amino acid or dna sequence dna identity usually, it has similar secondary and tertiary structure.Shared constitutional features makes the newcomer of gene family be identified and make equally non-natural method of amino-acids and the composition described to be used similarly easily herein.Suppose the structural homology degree between the member of GH supergene family, can use the present invention that non-naturally encoded amino acid is incorporated among any member of GH supergene family.Each member of described protein families comprises 4 helical bundles.
Comprise G-CSF (people such as Zink, FEBS Lett.314:435 (1992); People such as Zink, Biochemistry 33:8453 (1994); People such as Hill, Proc.Natl.Acad.Sci.USA 90:5167 (1993)), GM-CSF (Diederichs, people Science 154:1779-1782 (1991) such as K.; Walter et al, J.Mol.Biol.224:1075-1085 (1992)), IL-2 (Bazan, J.F. and McKay, D.B.Science 257:410-413 (1992)), IL-4 (people such as Redfield, Biochemistry 30:11029-11035 (1991); People such as Powers, Science 256:1673-1677 (1992)) and IL-5 (people such as Milburn, Nature 363:172-176 (1993)) although structure of a large amount of cytohormones is determined by X-ray diffraction and NMR research and is lacked significant elementary sequence homology, shows the surprising conservative of GH structure.Based on modeling and other research, IFN is considered to member (people such as Lee, the J.InterferonCytokine Res.15:341 (1995) of described family; People such as Murgolo, Proteins 17:62 (1993); People such as Radhakrishnan, Structure 4:1453 (1996); People such as Klaus, J.Mol.Biol.274:661 (1997)).Based on modeling and mutation research, EPO is considered to member (people such as Boissel, the J.Biol.Chem.268:15983-15993 (1993) of described family; People such as Wen, J.Biol.Chem.269:22839-22846 (1994)).Think that now above-mentioned all cells hormone and somatomedin constitute a big gene family.
Except that shared similar secondary and tertiary structure, the shared character of the member of described family must be with the cell surface receptor oligomerization with signal path in the active cells for it.Some GH family members that include, but is not limited to GH and EPO are in conjunction with the acceptor of single type and make it form homodimer.Include, but is not limited to other family members of IL-2, IL-4 and IL-6, in conjunction with the acceptor of more than one types and make described acceptor form heterodimer or high-order aggregate (people such as Davis, (1993), Science 260:1805-1808; People such as Paonessa, (1995), EMBO is J.14:1942-1951; Mott and Campbell, Current Opinion in Structural Biology 5:114-121 (1995)).Mutation research is showed, as GH, described other cytohormone and somatomedin contains a plurality of receptor binding sites, be generally 2 receptor binding sites, and in regular turn in conjunction with its homoreceptor (Mott and Campbell, Current Opinionin Structural Biology 5:114-121 (1995); People such as Matthews, (1996) Proc.Natl.Acad.Sci.USA93:9471-9476).As GH, described other family member's primary receptor combining site at first appears in 4 α spirals and the A-B ring.Specific amino acid in the helical bundle of participation receptors bind is different between the kinsfolk.With relevant on the interactional most of cell surface receptor structure of the member of GH supergene family and comprise second largest multigene family.Referring to (for example), United States Patent (USP) the 6th, 608, No. 183, it is to incorporate this paper by reference into.
The common conclusions that obtains from each member's of GH supergene family mutation research is, do not trend towards usually being involved in the receptors bind in conjunction with the ring of α spiral.As if specifically, lack the B-C ring is unnecessary to great majority (if not all) family member's receptors bind.Therefore, the B-C ring can be by the non-naturally encoded aminoacid replacement of describing herein of pressing among the member of GH supergene family.The aminoacid replacement that A-B ring, C-D ring (with the Interferon, rabbit/IL-10 sample member's of GH superfamily D-E ring) also can be existed by non-natural.Also trend towards not being involved in the receptors bind and also can be to be used to introduce the amino acid whose position that non-natural exists near the amino acid of spiral A with away from the amino acid of last spiral.In certain embodiments, non-naturally encoded amino acid is that any position in the preceding amino acid whose ring structure more than 1,2,3,4,5,6,7 or 7 that includes, but is not limited to A-B, B-C, C-D or D-E ring replaces.In certain embodiments, one or more non-naturally encoded amino acid is to locate to replace in the back amino acid more than 1,2,3,4,5,6,7 or 7 of A-B, B-C, C-D or D-E ring.
Some member who includes, but is not limited to the GH family of EPO, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10, IL-12p35, IL-13, IL-15 and interferon-is contained the sugar that N-is connected and/or O-connects.Glycosylation position in the protein almost appears in the ring zone and not in the α helical bundle specially.Because ring zone is not involved in the receptors bind usually and because it is the covalently bound position that is used for glycosyl group, so the efficient part of its may to be the aminoacid replacement that is used for that non-natural is existed introduce protein.In protein, comprise the position that N-connects the naturally occurring aminoacid replacement of amino acid possibility right and wrong that is connected the glycosylation position with O-, expose because described amino acid is the surface.Therefore, native protein mass-energy allows that being connected in proteinic huge glycosyl group and glycosylation position at described position trends towards locating away from receptor binding site.
May be other members that find in the future the GH supergene family.The newcomer of GH supergene family can pass through the area of computer aided secondary of the protein sequence predicted and tertiary structure analysis and discern in order to the selection technology that identification is incorporated into the molecule of concrete target by design.The member of GH supergene family has 4 or 5 usually and passes through non-helical shape amino acid (ring zone) bonded amphipathic helix.Protein can contain the hydrophobicity signal sequence to promote the secretion from cell at its N-end.The member of the described GH supergene family of finding afterwards is also included among the present invention.Relevant application case is to be disclosed as the international application of the title of WO 05/074650 for " Modified Four Helical BundlePolypeptides and Their Uses " on August 18th, 2005, and it is to incorporate this paper by reference into.
Therefore, provide the description of hGH to be used for the illustrative purpose and only illustrate, and not as the restriction of the category of method, composition, strategy and the technology described herein.In addition, mention that in the application's case the hGH polypeptide is intended to use the common name as the example of any tethelin.Therefore, should be appreciated that herein any member that the modification described about hGH polypeptide or protein and chemical action can similarly be applicable to the GH supergene family, comprise the described member who specifically lists in herein.
III. select codon
The genetic code subframe of selection codon expansion protein biosynthesizing device of the present invention.For example, select codon to include, but is not limited to 3 unique base codons, nonsense codon, such as the terminator codon that includes, but is not limited to amber codon (UAG), ocher codon or opal codon (UGA), the non-natural codon, 4 or 4 above base codons, rare codon or the like.Concerning the those skilled in the art, it is evident that, there is broad range in the quantity that can be introduced into the selection codon in desired gene or the polynucleotide, include, but is not limited in the single polynucleotide of at least a portion of coding hGH polypeptide, have the selection codon more than 1 or 1, more than 2 or 2, more than 3 or 3, more than 4,5,6,7,8,9,10 or 10.
In one embodiment, method relates to using selects codon, and it is for being used to incorporate into the terminator codon of one or more alpha-non-natural amino acids in vivo.For example, produce the O-tRNA that identification includes, but is not limited to the terminator codon of UAG, and by O-RS with desired alpha-non-natural amino acid amino acidylate.Described O-tRNA can not distinguish by naturally occurring host's aminoacyl-tRNA synthetase.Habitual positional mutation can be introduced the terminator codon that includes, but is not limited to TAG in order to the position of being paid close attention in the polypeptide of being paid close attention to.Referring to (for example), Sayers, people such as J.R. (1988), 5 '-3 ' Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res,16:791-802.When the nucleic acid of the polypeptide of paying close attention to when O-RS, O-tRNA and coding in vivo made up, response UAG codon was incorporated the polypeptide that alpha-non-natural amino acid obtains containing at prescribed position alpha-non-natural amino acid into.
Can finish incorporating into of alpha-non-natural amino acid in vivo and can significantly not disturb eukaryotic host cell.For example, because the inhibition effect of UAG codon decide on the competition between the O-tRNA that includes, but is not limited to amber suppressor tRNA and the eucaryon releasing hormone (including, but is not limited to eRF) (it is incorporated into terminator codon and peptide of causing in the growth discharges from rrna), can regulate by the expression level of (including, but is not limited to) increase O-tRNA and/or suppressor gene tRNA so suppress effect.
Alpha-non-natural amino acid also can be encoded by rare codon.For example, when the arginine concentration in vitro protein synthesis reacts reduces, verified rare arginine codon, promptly AGG is effective concerning insert Ala by the synthetic tRNA by the L-Ala acidylate.Referring to (for example), people such as Ma, Biochemistry,32:7939 (1993).Under described situation, synthetic tRNA and the naturally occurring tRNAArg competition that is present in as less important species in the colon bacillus.Some organisms are not used all triplet codons.Unspecified codon AGA in the micrococcus luteus (Micrococcus luteus) has been applied to inserting amino acid in the transcription/translation extract in vitro.Referring to (for example), Kowal and Oliver, Nucl.Acid.Res.,25:4685 (1997).Can produce component of the present invention to use described rare codon in vivo.
Select codon also to comprise expansion cipher that includes, but is not limited to 4 or 4 above base codons, such as 4,5,6 or 6 above base codons.The example of 4 base codons includes, but is not limited to AGGA, CUAG, UAGA, CCCU, and is like that.The example of 5 base codons includes, but is not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC, and is like that.Feature of the present invention comprises that expansion cipher is used in inhibition based on frameshit.4 or 4 above base codons can (include, but is not limited to) one or more alpha-non-natural amino acids and insert in same protein.For example, have anticodon loop, the sudden change O-tRNA that includes, but is not limited to special frame shift suppressor tRAN that for example has 8-10nt anticodon loop at least exists down, and 4 or 4 above base codons can be read as single amino acid.In other embodiments, anticodon loop decodable code (including, but is not limited to) 4-base codon, 5-base codon or 6-base codon or more than at least 6-base codon at least at least at least.Because there are 256 kinds of possible 4-base codons, so can in same cell, use 4 or 4 above base codons a plurality of alpha-non-natural amino acids of encoding.Referring to, people such as Anderson, (2002) Exploringthe Limits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codonsand Identification of " Shifty " Four-base Codons with a Library Approach in Escherichia coli J.Mol.Biol.307:755-769.
For example, use in vitro biosynthetic means, 4-base codon has been used to alpha-non-natural amino acid is incorporated in the protein.Referring to (for example), people such as Ma, (1993) Biochemistry, 32:7939; With people such as Hohsaka, (1999) J.Am.Chem.Soc,121:34.CGGG and AGGU are in order in vitro to incorporate the NBD derivative of 2-naphthyl L-Ala and Methionin in the streptavidin into two frame shift suppressor tRNA through chemical acylation simultaneously.Referring to (for example), people such as Hohsaka, J.Am.Chem.Soc, 121:12194.In vivo in the research, people such as Moore have studied the ability that the tRNALeu derivative with NCUA anticodon suppresses UAGN codon (N can be U, A, G or C), and find that tetrad UAGA can be by having the tRNALeu of UCUA anticodon, efficient decoding with 13 to 26%, decoding on a small quantity in 0 or-1 framework simultaneously.Referring to, people such as Moore, J.Mol.Biol.,298:195.In one embodiment, can be used among the present invention based on expansion cipher of rare codon or nonsense codon, it can reduce missense at other unwanted positions and read with frameshit and suppress.
Concerning given system, select codon also can comprise one of natural 3 base codons, wherein the endogenous system does not use (or seldom using) natural base codon.For example, described system comprises that shortage can pick out the system of the tRNA of natural 3 base codons, and/or wherein 3 base codons are systems of rare codon.
Select codon optionally to comprise the non-natural base pair.Described non-natural base pair further expands existing genetic alphabet table.An extra base pair makes the number of triplet codon be increased to 125 from 64.The character of the 3rd base pair comprise stable and optionally base pairing, with high frequency high fidelity by polysaccharase effectively enzymatic incorporate among the DNA and effective successive primer extension in the synthetic back of nascent non-natural base pair.The description that can be suitable for the non-natural base pair of method and composition comprises people such as (for example) Hirao, (2002) An unnatural base pair for incorporating aminoacid analogues into protein, Nature Biotechnology, 20:177182.Again referring to, Wu, people such as Y., (2002) J.Am.Chem.Soc.124:14626-14630.Other relevant open cases are listed in hereinafter.
Concerning in vivo using, the non-natural nucleoside permeable membrane and through phosphorylation to form corresponding triphosphate.In addition, the genetic information of increase is stable and can not be destroyed by cellular enzymes.Benner and other people's previous effort utilization is different from the hydrogen bond pattern of the described pattern of typical Watson-Crick centering, and wherein the most noticeable example is that iso-C:iso-G is right.Referring to (for example), people such as Switzer, (1989) J.Am.Chem.Soc, 111:8322; With people such as Piccirilli, (1990) Nature.343:33; Kool, (2000) Curr.Opin.Chem.Biol.,4:602.Described base in general to a certain extent with natural base mispairing and can not duplicate enzymatic.Kool and colleague's proof, the hydrophobicity packing interaction energy displacement hydrogen bond between the base is to drive the formation of base pair.Referring to, Kool, (2000) Curr.Opin.Chem. Biol.,4:602; With Guckian and Kool, (1998) Angew.Chem.Int.Ed.Engl., 36,2825.In the process of being devoted to develop the non-natural base pair that satisfies all above-mentioned requirements, Schultz, Romesberg and colleague are systematically synthetic and studied a series of non-natural hydrophobicity bases.It is more stable to find that PICS:PICS self contrasts natural base pair, and can be effectively crin promise fragment by the colon bacillus dna polymerase i (Klenow fragment KF) incorporates among the DNA.Referring to (for example), people such as McMinn, (1999) J.Am.Chem.Soc,121:11585-6; With people such as Ogawa, (2000) J.Am.Chem.Soc, 122:3274.3MN:3MN self synthesizes with the efficient and the selectivity that enough are used for biological function passing through KF.Referring to (for example), people such as Ogawa, (2000) J.Am.Chem.Soc,122:8803.Yet two bases are taken on the chain terminator that further duplicates.Recently, having developed can be in order to duplicate the right mutation DNA polymerase of PICS self.In addition, reproducible 7AI self is right.Referring to (for example), people such as Tae, (2001) J.Am.Chem.Soc, 123:7439.Also developed novel metal base pair, i.e. Dipic:Py, its form in conjunction with copper (II) back stable right.Referring to, people such as Meggers, (2000) J.Am.Chem.Soc,122:10714.Because expansion cipher and non-natural codon in essence with natural codon quadrature, so method of the present invention can utilize described character to produce its orthogonal tRNA.
Translating bypath system also can be in order to incorporate alpha-non-natural amino acid in the desired polypeptide into.In translating bypath system, big sequence is merged in the gene but is not translated into protein.Sequence contains to serve as induces rrna to skip sequence and the structure of the inset that recovers in the downstream of inset to translate.
In certain embodiments, protein of being paid close attention in method of the present invention and/or the composition or polypeptide (or its part) are to encode by nucleic acid.Usually, nucleic acid comprises at least 1 selection codon, at least 2 selection codons, at least 3 selection codons, at least 4 selection codons, at least 5 selection codons, at least 6 selection codons, at least 7 selection codons, at least 8 selection codons, at least 9 selection codons, the selection codon more than 10 or 10.
The protein that coding is paid close attention to or the gene of polypeptide can use method that the those skilled in the art knows and that describe to suddenly change to comprise that (for example) one or more selection codon is used to incorporate into alpha-non-natural amino acid herein.For example, the proteinic nucleic acid of being paid close attention to is selected codon through sudden change to comprise one or more, provides to incorporate one or more alpha-non-natural amino acids into.The present invention includes any described variant, it includes, but is not limited to mutant, and (for example) comprises any proteinic version of at least a alpha-non-natural amino acid.Similarly, the present invention also comprises corresponding nucleic acid, that is to say to have any nucleic acid of the selection codon of one or more one or more alpha-non-natural amino acids of encoding.
The proteinic nucleic acid molecule of being paid close attention to of coding such as hGH polypeptide can easily be introduced halfcystine through sudden change with any desired position at polypeptide.Halfcystine by widely in order to reactive molecule, water-soluble polymers, protein or multiple other molecule are incorporated into paid close attention to protein on.Be suitable for incorporating halfcystine in the desired position of polypeptide method for known to the those skilled in the art, such as the described method and the standard mutating technology that are described in No. the 6th, 608,183, the United States Patent (USP) incorporating this paper by reference into.
IV. non-naturally encoded amino acid
The non-naturally encoded amino acid of utmost point extensive farming class is applicable to the present invention.Any amount of non-naturally encoded amino acid can be introduced in the hGH polypeptide.In general, with respect to 20 kinds of amino acid common, genetic coding (just, L-Ala, arginine, asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, L-iLeu, L-LEU, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan), the non-naturally encoded amino acid through introducing is substantially chemically inert.In certain embodiments, non-naturally encoded amino acid comprise effectively and optionally with do not see 20 kinds of functional groups' (including, but is not limited to azido-, ketone, aldehyde and amino oxygen base) reactions in the common amino acid to form the side chain functionalities of stable binding substances.
The universal architecture of a-amino acid is described as follows (formula I):
Figure A20058004446300381
Non-naturally encoded amino acid is generally any structure with above listed formula, and wherein the R group is to be different from used substituent any substituting group in 20 kinds of natural amino acids, and goes for the present invention.Because non-naturally encoded amino acid of the present invention only is different from natural amino acid usually on the structure of side chain, so non-naturally encoded amino acid forms amido linkage with including, but is not limited to natural or non-naturally encoded amino acid whose other amino acid, described amido linkage is to form with its same manner that forms in naturally occurring polypeptide.Yet non-naturally encoded amino acid has the side-chain radical that itself and natural amino acid can be distinguished.For example, R optionally comprise alkyl-, aryl-, acyl group-, ketone group-, azido-, hydroxyl-, hydrazine, cyano group-, halogen-, hydrazides, thiazolinyl, alkynyl, ether, mercaptan, seleno-, alkylsulfonyl-, boronate, tenth of the twelve Earthly Branches friend's acidic group, phosphate, phosphono, phosphine, heterocyclic radical, ketenes, imines, aldehyde, ester, thioic acid sulfoacid, azanol, amino or the like or its any combination.But include, but is not limited to comprise the amino acid of the linking agent of photoactivation applicable to the amino acid that the non-natural that other are paid close attention to of the present invention exists, amino acid through spin labeling, fluorescigenic amino acid, the amino acid of bond, metallic amino acid, radioactivity amino acid, amino acid with novel functional group, covalently or non-covalentlyly with the amino acid of other interactions of molecules, photic shrouding and/or the amino acid of intramolecular photosensitization, the amino acid that comprises vitamin H or vitamin H analogue, such as through sugar-substituted Serine through glycosylated amino acid, amino acid through other carbohydrate modifications, the amino acid that contains ketone, the amino acid that comprises polyoxyethylene glycol or polyethers, amino acid through the heavy atom replacement, but the amino acid of chemical cracking and/or photo-cleavage, the amino acid that has the elongation side chain when with the natural amino acid comparison, described elongation side chain include, but is not limited to polyethers or (including, but is not limited to) greater than about 5 or greater than the long chain hydrocarbon of about 10 carbon, the glycoprotein amino acid that contains through the carbon connection, the amino acid of redox active, contain the amino acid of amino thioic acid sulfoacid and comprise one or more toxicity amino acid partly.
Go for the present invention and be applicable to that exemplary non-naturally encoded amino acid with water-soluble polymers reaction includes, but is not limited to have carbonyl, amino oxygen base, hydrazine, hydrazides, Urea,amino-, the described amino acid of trinitride and alkyne reaction group.In certain embodiments, non-naturally encoded amino acid comprises the carbohydrate part.Described amino acid whose example comprises N-ethanoyl-L-glucosamine base-L-Serine, N-ethanoyl-L-galactosaminyl-L-Serine, N-ethanoyl-L-glucosamine base-L-Threonine, N-ethanoyl-L-glucosamine base-altheine and O-epichitosamine base-L-Serine.Described amino acid whose example comprises also that wherein the naturally occurring N-key between the amino acid and carbohydrate or O-key are by uncommon covalent linkage on the nature-include, but is not limited to alkene, oxime, thioether, acid amides, suchlike covalent linkage metathetical example.Described amino acid whose example also comprises the carbohydrate that is not common in the naturally occurring protein, such as 2-deoxidation-glucose, 2-deoxy-galactose or the like.
The many non-naturally encoded amino acid that provides herein can available from (for example) Sigma-Aldrich (St.Louis, MO, USA), Novabiochem (a division of EMD Biosciences, Darmstadt, Germany) or Peptech (Burlington, MA, USA).The described amino acid that can not buy be optionally by provided herein or use to the standard method known to the those skilled in the art and synthesize.Concerning organic synthesis technology, referring to (for example), Fessendon and Fessendon's Organic Chemistry(1982, second edition, Willard Grant Press, BostonMass.); March's Advanced Organic Chemistry(third edition, 1985, Wiley and Sons, New York); With Carey and Sundberg Advanced Organic Chemistry(third edition, part A and B, 1990, PlenumPress, New York).Referring to, U.S. Patent Application Publication case 2003/0082575 and 2003/0108885, it all incorporates this paper by reference into again.Except that the alpha-non-natural amino acid that contains novel side chain, go for alpha-non-natural amino acid of the present invention and also optionally comprise modified backbone structure, include, but is not limited to be illustrated by the structure of formula II and III:
Figure A20058004446300391
Wherein Z comprises OH, NH usually 2, SH, NH-R ' or S-R '; Can be identical or different X and Y and comprise S or O usually, and optionally normally be selected from above identical inventory and hydrogen the composition of the described R group of the alpha-non-natural amino acid with formula I for identical or different R and R '.For example, alpha-non-natural amino acid of the present invention optionally comprises as by the illustrated amino of formula II and III or the replacement of carboxyl.The alpha-non-natural amino acid of described type includes, but is not limited to alpha-hydroxy acid, α-thioic acid sulfoacid, alpha-amino group carbothioic acid ester, includes, but is not limited to have corresponding to the side chain of 20 kinds of common natural amino acids or those alpha-non-natural amino acids of non-natural side chain.In addition, the replacement at the alpha-carbon place optionally includes, but is not limited to such as D-L-glutamic acid, D-L-Ala, D-methyl-O-tyrosine, the suchlike L of aminobutyric acid, D or α-α-dibasic amino acid.Other structure surrogates comprise the cyclic amino acid such as proline analogs and 3,4,6,7,8 and 9 yuan of loop proline analogues, such as the β and the γ amino acid of Beta-alanine that is substituted and γ-An Jidingsuan.
Many alpha-non-natural amino acids are based on such as tyrosine, glutamine, phenylalanine, suchlike natural amino acid, and be applicable to the present invention.The tyrosine analogue includes, but is not limited to; the tyrosine that a tyrosine that the tyrosine of para-orientation, ortho position replace and a position replace, the tyrosine that wherein is substituted comprises (including, but is not limited to) ketone group (including, but is not limited to ethanoyl), benzoyl, amino, hydrazine, azanol, thiol group, carboxyl, sec.-propyl, methyl, C 6-C 20Straight or branched hydrocarbon, saturated or unsaturated hydrocarbons, O-methyl, polyether-based, nitro, alkynyl or the like.In addition, also contain through polysubstituted aryl rings.Go for glutamine analogue of the present invention and include, but is not limited to, the derivative of Alpha-hydroxy derivative, γ-replacement, cyclic derivatives and the glutamine derivative that replaces through acid amides.Go for example phenylalanine analogues of the present invention and include, but is not limited to the phenylalanine of para-orientation, the phenylalanine of ortho position replacement and the phenylalanine that a position replaces, wherein substituting group comprises (including, but is not limited to) hydroxyl, methoxyl group, methyl, allyl group, aldehyde, azido-, iodo, bromo, ketone group (including, but is not limited to ethanoyl), benzoyl, alkynyl or the like.The specific example that goes for alpha-non-natural amino acid of the present invention includes, but is not limited to ethanoyl-L-phenylalanine; O-methyl-L-tyrosine; L-3-(2-naphthyl) L-Ala; the 3-methylphenylalanine; O-4-allyl group-L-tyrosine; 4-propyl group-L-tyrosine; three-O-ethanoyl-GlcNAc β-Serine; L-Dopa; fluoridize phenylalanine; sec.-propyl-L-phenylalanine; to azido--L-phenylalanine; to acyl group-L-phenylalanine; to benzoyl-L-phenylalanine; the L-phosphoserine; the phosphono Serine; phosphono tyrosine; to the iodo-phenylalanine; to bromophenyl alanine; to amino-L-phenylalanine; sec.-propyl-L-phenylalanine and to propargyloxy-phenylalanine, like that.The example that goes for the structure of various alpha-non-natural amino acids of the present invention is provided in (for example) title among the WO 2002/085923 of " In vivoincorporation of unnatural amino acids ".Also can be concerning other methionine(Met) analogues referring to, people such as Kiick, (2002) Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligation, PNAS99:19-24, it is to incorporate this paper by reference into.
The composition of the hGH polypeptide that comprises alpha-non-natural amino acid (such as to (propargyloxy)-phenylalanine) is provided in one embodiment.Also provide and comprise (propargyloxy)-phenylalanine and include, but is not limited to protein and/or the various compositions of cell.On the one hand, comprise that the composition to (propargyloxy)-phenylalanine alpha-non-natural amino acid comprises orthogonal tRNA in addition.Alpha-non-natural amino acid can (include, but is not limited to covalently) be binding on orthogonal tRNA; it includes, but is not limited to covalently be binding on orthogonal tRNA by amino-acyl bond, covalently is binding on 3 ' OH of terminal ribose of orthogonal tRNA or 2 ' OH or the like.
The advantage and the manipulation of range protein are provided via the chemical part that can incorporate the alpha-non-natural amino acid in the protein into.For example, the reactivity of the uniqueness of ketone allows by many any reagent that contain hydrazine or contain azanol reagent modifying protein optionally in vitro and in vivo.Heavy atom alpha-non-natural amino acid (for example) is applicable to phasing x-ray structure data.Use the site specific of the heavy atom of alpha-non-natural amino acid to introduce selectivity and the adaptability that selected location also is provided as heavy atom.Photoreactivity alpha-non-natural amino acid (including, but is not limited to have the amino acid of benzophenone and aromatic yl azide (including, but is not limited to azidomethyl phenyl nitrogenize thing) side chain), (for example) are allowed proteinic effectively in vivo and in vitro photocrosslinking.The example of photoreactivity alpha-non-natural amino acid includes, but is not limited to azido--phenylalanine with to benzoyl-phenylalanine.Protein with photoreactivity alpha-non-natural amino acid can be therefore optionally crosslinked by the exciting of photoreactive group that Instantaneous Control is provided.In an example, the methyl of alpha-non-natural amino acid can be through isotope-labeled (including, but is not limited to) methyl substituted, as (including, but is not limited to) local structure and the dynamic (dynamical) probe for nucleus magnetic resonance and vibrational spectroscopy use.Alkynyl or azido-functional group (for example) allow by [3+2] cycloaddition reaction, with molecular selectivity ground modifying protein.
Incorporating non-natural amino acid in the polypeptide at N-terminal can be included as and be different from 20 kinds of natural amino acids used substituent any substituent R group and be different from the NH that normally is present in the a-amino acid (referring to formula I) 2Second reactive group of group.Similar alpha-non-natural amino acid can be incorporated into to have and is different from second reactive group that normally is present in the COOH group in the a-amino acid (referring to formula I) at C-terminal.
Alpha-non-natural amino acid of the present invention can be through selecting or designing to be provided at unavailable other features in 20 kinds of natural amino acids.For example, alpha-non-natural amino acid can be optionally through design or select that it incorporates wherein proteinic biological property into to change (for example).For example, following character can optionally change by alpha-non-natural amino acid is contained in the protein: decompose in the toxicity, body, solubility, for example thermostability, stability to hydrolysis, oxidative stability, the suchlike stability of the short degraded of antienzyme, the easiness of purifying and handling, textural property, spectral quality, the chemistry and/or actinism, catalytic activity, redox potential, transformation period, with other molecules (for example) covalently or the ability of non-covalently reacting, like that.Incorporate No. the 11/046th, 432, the U.S. patent application case of this paper by reference into, many different non-naturally encoded amino acid have been discussed.
The chemosynthesis of alpha-non-natural amino acid
Be applicable to many alpha-non-natural amino acids of the present invention be can available from Sigma (USA) or Aldrich (Milwaukee, WI, USA).The described amino acid that can not buy be optionally by provided herein or by provided in the various open cases or use to the standard method known to the those skilled in the art and synthesize.About organic synthesis technology, referring to (for example), Fessendon and Fessendon's Organic Chemistry(1982, second edition, Willard Grant Press, Boston Mass); March's Advanced Organic Chemistry(third edition, 1985, Wiley and Sons, NewYork); With Carey and Sundberg Advanced Organic Chemistry(third edition, part A and B, 1990, Plenum Press, New York).Other open cases of synthetic of describing alpha-non-natural amino acid comprise that (for example) title is the WO 2002/085923 of " In vivo incorporation of Unnatural Amino Acids "; People such as Matsoukas, (1995) J .Med.Chem.,38,4660-4669; King, F.E.﹠amp; Kidd, D.A.A. (1949) A New Synthesis ofGlutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates. J.Chem. Soc.,3315-3319; Friedman, O.M.﹠amp; Chatterrji, R. (1959) Synthesis of Derivatives ofGlutamine as Model Substrates for Anti-Tumor Agents. J.Am.Chem.Soc.81,3750-3752; Craig, people such as J.C. (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-l-methylbutyl] amino] quinoline (Chloroquine). J.Org.Chem.53,1167-1170; Azoulay, M., Vilmont, M. ﹠amp; Frappier, F. (1991) Glutamine analogues asPotential Antimalarials, Eur.J.Med.Chem.26,201-5; Koskinen, A.M.P.﹠amp; Rapoport, H. (1989) Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues. J.Org.Chem.54,1859-1866; Christie, B.D.﹠amp; Rapoport, H. (1985) Synthesis ofOptically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization. J.Org. Chem.50:1239-1246; People such as Barton, (1987) Synthesis of Novel alpha-Amino-Acids andDerivatives Using Radical Chemistry:Synthesis of L-and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid and Appropriate Unsaturated Derivatives. Tetrahedron43:4297-4308; With people such as Subasinghe, (1992) Quisqualic acid analogues:synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized stte. J.Med.Chem.35:4602-7.Also can be No. 2004/0198637, the U.S. Patent Publication case US of " Protein Arrays " referring to title, it be to incorporate this paper by reference into.
For example, ethanoyl-(+/-)-phenylalanine and an ethanoyl-(+/-)-phenylalanine synthetic is described in people such as Zhang, Z., among the Biochemistry 42:6735-6746 (2003), it is to incorporate this paper by reference into.Other contain carbonylamino acid and can similarly be prepared by the those skilled in the art.The carbonyl functional group can be under mild conditions, in the aqueous solution optionally with contain hydrazine, contain hydrazides, contain azanol or contain Urea,amino-reagent react to be respectively formed at corresponding hydrazone stable under the physiological condition, oxime or semicarbazone key.Referring to (for example), Jencks, W.P., J.Am.Chem.Soc.81,475-481 (1959); Shao, J.and Tam, J.P., J.Am.Chem.Soc.117:3893-3899 (1995).In addition, unique reactivity of carbonyl is allowed the selective modification in the presence of other amino acid side chains.Referring to (for example), Cornish, people such as V.W., J.Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F.﹠amp; Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, people such as L.K., Science 276:1125-1128 (1997).
The biosynthesizing of alpha-non-natural amino acid
Many biosynthesizing path Already in is used to produce amino acid and other compounds in the cell.Though the biosynthetic means of concrete alpha-non-natural amino acid may not be present in fact in (including, but is not limited to) cell, the invention provides described method.For example, the biosynthesizing path of alpha-non-natural amino acid produces by increasing novel enzyme or changing existing host cell path in host cell.Other novel enzymes optionally are the enzyme of naturally occurring enzyme or artificial exploitation.For example, the biosynthesizing of p-Aminophenylalanine (is to present among the WO 2002/085923 of " In vivoincorporation of unnatural amino acids " as title) is fixed against the combination of interpolation from the known enzyme of other biological body.The gene of described enzyme can be by introducing in the eukaryotic cell cell transition with the plasmid that comprises gene.When expressing in cell, gene provides the enzymatic path with synthetic desired compound.Optionally the example of the type of the enzyme of Tian Jiaing is provided in hereinafter the example.Other enzyme sequence is found in (for example) gene pool.Also optionally add in the cell with the enzyme of the same manner with manually exploitation.So, the resource of manipulated cell device and cell is produced alpha-non-natural amino acid.
The whole bag of tricks can be used for producing the enzyme that uses or be used to develop the novelty of existing route for the biosynthesizing path.For example, optionally will (include, but is not limited to) as by Maxygen, the recursiveness reorganization (can buy on the maxygen.com at World WideWeb) of Inc. exploitation is used to develop novel enzyme and path.Referring to (for example), Stemmer (1994), Rapidevolution of a protein in vitro by DNA shuffling, Nature370 (4): 389-391; And Stemmer, (1994), DNA shuffling by random fragmentation and reassembly:In vitro recombination formolecular evolution, Proc.Natl.Acad.Sci.USA., 91:10747-10751.Similarly, optionally will be by the DesignPath of Genencor exploitation TM(can buy on the genencor.com at World Wide Web) is used for the metabolic pathway through engineering approaches, includes, but is not limited to be used for the through engineering approaches path to produce O-methyl-L-tyrosine at cell.Described technology is used the novel gene that (including, but is not limited to) discern by functional genome and the combination of molecular evolution and design, and rebuilds existing route in host organisms.Diversa Corporation (can buy on the diversa.com at World Wide Web) also provides the rapid screening routine library (including, but is not limited to) in gene and gene path to produce the technology in novel path.
Usually, alpha-non-natural amino acid by the production of through engineering approaches biosynthesizing of the present invention path, be enough to be used for the biosynthesizing of effective protein proteins matter, do not produce but do not reach the concentration that influences other amino acid whose concentration or exhaust the degree of cell resource with what include, but is not limited to the n cell amount.The typical concentration of in vivo producing is that about 10mM is to about 0.05mM in this way.Ribosomal protein synthesizes and the production of the alpha-non-natural amino acid of cell growth in case cell by the plasmid transition that comprises in order to the gene of producing the required enzyme of particular path and alpha-non-natural amino acid, is in vivo selected just optionally to be used in order to further optimization.
The cell of alpha-non-natural amino acid absorbs
It is a problem of being considered in design with when selecting (including, but is not limited to) to be used for incorporating into the alpha-non-natural amino acid of protein usually that the alpha-non-natural amino acid of cell absorbs.For example, the high charge density of a-amino acid hints that described compound can not be for permeable cell.Natural amino acid is to absorb in the eukaryotic cell by many haulage system based on protein.Can carry out rapid screening, it evaluates which kind of alpha-non-natural amino acid (if any) is absorbed by cell.Referring to (for example), (for example) title is the toxicological detection among No. 2004/0198637, the U.S. Patent Publication case US of " Protein Arrays ", and described patent is to incorporate this paper by reference into; And Liu, D.R.﹠amp; Schultz, P.G. (1999) Progress toward the evolution of an organism with an expanded genetic code. PNAS United StatesToxicological detection among the 96:4780-4785.Though absorb and to analyze with various calibratings easily, another that designs the alpha-non-natural amino acid that stands cell absorption path is chosen as and provides the biosynthesizing path to produce amino acid in vivo.
V. the polypeptide that has alpha-non-natural amino acid
Can be owing to the various purposes that include, but is not limited to following purpose are carried out incorporating into of alpha-non-natural amino acid: regulate the change of protein structure and/or function, change size, acidity, nucleophilicity, hydrogen bond, hydrophobicity, the accessibility of proteolytic enzyme target site, with a part as target (include, but is not limited to protein arrange part), add bioactive molecules, connect polymkeric substance, connect radionuclide, regulate serum half-life, regulate tissue penetration rate (for example tumour), regulate active transport, regulate tissue, immunogenicity is regulated in cell or organ specificity or distribution, regulates proteolytic enzyme resistibility or the like.The albumen mass-energy that comprises alpha-non-natural amino acid has enhanced or even brand-new clever catalytic property or biophysical properties.For example, following character optionally changes by alpha-non-natural amino acid is contained in the protein: decomposition in the toxicity, body, textural property, spectral quality, chemistry and/or actinism, catalytic capability, transformation period (including, but is not limited to serum half-life), with other molecules (including, but is not limited to) covalently or the ability of non-covalently reacting, like that.Comprise that the proteinic composition that comprises at least a alpha-non-natural amino acid is applicable to the research of therapy, diagnosis, katalaze enzyme, industrial enzyme, conjugated protein (including, but is not limited to antibody) and (including, but is not limited to) protein structure and function that (including, but is not limited to) is novel.Referring to (for example), Dougherty, (2000) UnnaturalAmino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4:645-652.
In one aspect of the invention, composition comprises that at least a kind has at least a kind, the protein of (including, but is not limited to) alpha-non-natural amino acid more than at least 2 kinds, at least 3 kinds, at least 4 kinds, at least 5 kinds, at least 6 kinds, at least 7 kinds, at least 8 kinds, at least 9 kinds or at least 10 kinds or 10 kinds.Alpha-non-natural amino acid can be identical or different, exists in (including, but is not limited to) protein to comprise a kind, 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds, 8 kinds, 1,2,3,4,5,6,7,8, the different position more than 9 or 10 or 10 of the different alpha-non-natural amino acid more than 9 kinds or 10 kinds or 10 kinds.On the other hand, composition comprises and is present in the protein that concrete amino acid whose at least a (but being less than all) in the protein replaces through alpha-non-natural amino acid.Have more than a kind of protein of alpha-non-natural amino acid given, that alpha-non-natural amino acid can be is identical or different (include, but is not limited to protein can comprise dissimilar alpha-non-natural amino acid more than 2 kinds or 2 kinds maybe can comprise 2 kinds of identical alpha-non-natural amino acids).Have more than the protein of 2 kinds of alpha-non-natural amino acids given, alpha-non-natural amino acid can be identical, different or is the combination of a plurality of alpha-non-natural amino acids with identical type alpha-non-natural amino acid different with at least a kind.
Protein of being paid close attention to at least a kind of alpha-non-natural amino acid or polypeptide are features of the present invention.The present invention also comprises having at least a kind of polypeptide or protein that uses the alpha-non-natural amino acid of the compositions and methods of the invention production.Vehicle (including, but is not limited to pharmaceutically acceptable vehicle) also can exist with protein.
Concerning produce the protein of being paid close attention to or polypeptide with at least a kind of alpha-non-natural amino acid in eukaryotic cell, protein or polypeptide will be translated modification after will generally including eucaryon.In certain embodiments, protein comprise at least a kind of alpha-non-natural amino acid and at least a kind by eukaryotic cell in vivo carry out after translate modification, translate after wherein to modify and non-ly undertaken by prokaryotic cell prokaryocyte.For example, after translate to modify and comprise that (including, but is not limited to) acetylize, acidylate, lipid-modified, palmitoylation effect, cetylate interpolation, phosphorylation, glycolipid key are modified, glycosylation, like that.On the one hand, after translate to modify and comprise by GlcNAc-asparagine key, oligosaccharides (is included, but is not limited to (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc) be connected to asparagine.Referring to table 1, its N-that has listed eukaryotic protein connects the example (also can have other the residue of not showing) of oligosaccharides.On the other hand, after translate to modify and comprise by GalNAc-Serine or GalNAc-Threonine key, or GlcNAc-Serine or GlcNAc-Threonine key, oligosaccharides (including, but is not limited to Gal-GalNAc, Gal-GlcNAc or the like) is connected to Serine or Threonine.
Table 1: the example of the oligosaccharides by the GLCNAC-bonding
Figure A20058004446300451
On the other hand, after translate and modify the proteolytic treatment comprise precursor, be assembled in many subelements protein or the macromole assembling, be translated to another position (include, but is not limited to be translated in the organoid such as endoplasmic reticulum, golgi body (Golgi apparatus), nuclear, lysosome, peroxysome, plastosome, chloroplast(id), vacuole or the like, or pass through the secretion path) in the cell.In certain embodiments, protein comprises secretion sequence or positioning sequence, epitope label, FLAG label, polyhistidyl label, GST syzygy or the like.The United States Patent (USP) the 4th, 963,495 and 6,436 of incorporating this paper by reference into No. 674, is described in detail through design and is constructed body with the excretory that improves the hGH polypeptide.
An advantage of alpha-non-natural amino acid is that its existence can be in order to add other chemical part of other molecules.Described modification can in vivo or in vitro be carried out in eucaryon or non-eukaryotic cell.Therefore, in certain embodiments, after translate that to modify be to finish by alpha-non-natural amino acid.For example, translating modification after can finish by nucleophilic-electrophilic reaction.Currently be used for the proteinic great majority reaction of selective modification and relate between nucleophilic and electrophilic reaction collocation thing and form covalent linkage, include, but is not limited to the reaction of α-halogen ketone and Histidine or cysteine side chain.Selectivity under the described situation is by the quantity of the nucleophilic residues in the protein and accessibility decision.In protein of the present invention, can use other more selectively to react, such as in vitro and in vivo, the reaction of non-natural ketone-amino acid and hydrazides or amino oxygen based compound.Referring to (for example), people such as Cornish, (1996) J.Am.Chem.Soc118:8150-8151; People such as Mahal, (1997) Science, 276:1125-1128; People such as Wang, (2001) Science292:498-500; People such as Chin, (2002) J.Am. Chem.Soc.124:9026-9027; People such as Chin, (2002) Proc.Natl.Acad.Sci.,99:11020-11024; People such as Wang, (2003) Proc Natl.Acad.Sci., 100:56-61; People such as Zhang, (2003) Biochemistry.42:6735-6746 with people such as Chin, (2003) Science, 301:964-7, all incorporate this paper by reference into for it.Described reaction allows with comprising that a large amount of reagent of fluorophore, linking agent, carbohydrate derivative and cytotoxic molecule come the almost any protein of selected marker.Be No. the 6th, 927,042, the United States Patent (USP) of " Glycoprotein Synthesis " referring to, title again, it is to incorporate this paper by reference into.(including, but is not limited to) translated modification after amino acid whose and also can be connected (including, but is not limited to use triaryl phosphine reagent) by Staudinger and carry out by azido-.Referring to (for example), people such as Kiick, (2002) Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligation, PNAS99:19-24.
The invention provides proteinic another high efficiency method of selective modification, its heredity that relates to alpha-non-natural amino acid is incorporated into.Can add proteinic molecule to includes, but is not limited to, derivative, resin, bead, second protein or the polypeptide (or more) of dyestuff, fluorophore, linking agent, carbohydrate derivative, polymkeric substance (including, but is not limited to the derivative of polyoxyethylene glycol), photocrosslinking agent, cytotoxin compounds, affinity labelling, vitamin H, polynucleotide (including, but is not limited to DNA, RNA or the like), metal chelator, cofactor, lipid acid, carbohydrate, like that.In one embodiment, described method further comprises to be incorporated alpha-non-natural amino acid in the protein into, and wherein alpha-non-natural amino acid comprises first reactive group; With make protein and the molecule that comprises second reactive group (include, but is not limited to mark, dyestuff, polymkeric substance, water-soluble polymers, the derivative of polyoxyethylene glycol, the photocrosslinking agent, radionuclide, cytotoxin compounds, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotides, water-soluble branch-shape polymer, cyclodextrin, inhibition Yeast Nucleic Acid, carbohydrate, biomaterial, nanoparticle, spin label, fluorophore, metallic part, radioactive segment, novel functional group, covalently or non-covalentlyly with the group of other interactions of molecules, the photic part of shrouding, but the part that actinic radiation excites, but the part of photoisomerization, vitamin H, the derivative of vitamin H, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, the side chain of elongation, sugar through the carbon connection, the redox activator, amino thioic acid sulfoacid, the toxicity part, isotope-labeled part, the biophysics probe, phosphorescent group, chemiluminescent group, the close group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable mark, small molecules, quantum dot, the nanometer transmitter, the radioactive nuleus thuja acid, the radioactivity transmitter, neutron capture agent, or any combination of above-mentioned above-mentioned substance or any other required compound or material) contact.
VI. comprise the in vivo generation of the amino acid whose hGH polypeptide of non-genetic coding
HGH polypeptide of the present invention can use modified tRNA and tRNA synthetic enzyme to add in vivo or replace not amino acids coding in naturally occurring system and produce.
Use the not method that is used to produce tRNA and tRNA synthetic enzyme of amino acids coding in naturally occurring system, be described in (for example) U.S. Patent Application Publication case 2003/0082575 the (the 10/126th, No. 927) and 2003/0108885 the (the 10/126th, 931) in, described patent is to incorporate this paper by reference into.Described method relates to and produces the transfer interpreter be independent of endogenic concerning the system of translating (and therefore be called sometimes " orthogonal ") synthetic enzyme and tRNA and work.Usually, the system of translating comprises orthogonal tRNA (O-tRNA) and orthogonal aminoacyl tRNA synthetase (O-RS).Usually, preferentially in the system of translating, the amino acid that exists with at least a non-natural picks out the selection codon that at least one can not be picked out by other tRNA in the system with amino acidylate of O-tRNA and O-tRNA to O-RS.Respond encoded selection codon, in the protein that the system that translates therefore produces non-naturally encoded aminoacid insertion in the system, thereby with in the position of amino acid " replacement " in the encoded polypeptide.
The multiple orthogonal tRNA and the aminoacyl tRNA synthetase that are used for concrete synthesizing amino acid is inserted into polypeptide are described in affiliated field, and are applicable to the present invention usually.For example, ketone-specificity O-tRNA/ aminoacyl-tRNA synthetase is described in Wang, people such as L., and Proc.Natl.Acad.Set USA 100:56-61 (2003) and Zhang, people such as Z. are among Biochem.42 (22): the 6735-6746 (2003).Exemplary O-RS or its part are to encode and comprise the aminoacid sequence that is disclosed in U.S. Patent Application Publication case 2003/0082575 and 2003/0108885 by polynucleotide sequence, and each patent is incorporated this paper by reference into.The O-tRNA molecule of the correspondence of using for O-RS also is described in U.S. Patent Application Publication case 2003/0082575 (the 10/126th, No. 927) and 2003/010888 (the 10/126th, No. 931), and described patent is to incorporate this paper by reference into.
The case description of trinitride-specificity O-tRNA/ aminoacyl-tRNA synthetase system is in Chin, and people such as J.W. are among the J.Am.Chem.Soc.124:9026-9027 (2002).Exemplary O-RS sequence to azido--L-Phe includes, but is not limited to, as U.S. Patent Application Publication case 2003/0108885 the (the 10/126th, No. 931) middle nucleotide sequence SEQ ID NO:14-16 and 29-32 and aminoacid sequence SEQ ID NO:46-48 and the 61-64 that discloses, described patent is to incorporate this paper by reference into.Be applicable to that exemplary O-tRNA sequence of the present invention includes, but is not limited to the nucleotide sequence SEQ ID NO:1-3 that discloses as in the U.S. Patent Application Publication case 2003/0108885 (the 10/126th, No. 931), described patent is to incorporate this paper by reference into.Concrete non-naturally encoded amino acid had other right case descriptions of specific O-tRNA/ aminoacyl-tRNA synthetase in U.S. Patent Application Publication case 2003/0082575 the (the 10/126th; No. 927) in, described patent is incorporated this paper by reference into.To contain the amino acid of ketone and contain O-RS and the O-tRNA that the amino acid of trinitride incorporates in the yeast saccharomyces cerevisiae (S.cerevisiae) and be described in Chin, people such as J.W. are among the Science 301:964-967 (2003).
Reported that some other quadratures are right.The glutaminyl that obtains from yeast saccharomyces cerevisiae tRNA and synthetic enzyme (referring to (for example), Liu, D.R. and Schultz, P.G. (1999) Proc.Natl.Acad.Sci.U.S.A.96:4780-4785), and aspartyl (referring to (for example), Pastrnak, people such as M., (2000) Helv.Chim.Acta83:2277-2286) and tyrosyl (referring to (for example), Ohno, people such as S., (1998) J.Biochem. (Tokyo.JpN.) 124:1065-1068; And Kowal, people such as A.K., (2001) Proc.Natl.Acad.Sci.U.S.A.98:2268-2273) system has been described and has been used for incorporating alpha-non-natural amino acid into intestinal bacteria potentially.From the intestinal bacteria glutaminyl (referring to (for example), Kowal, people such as A.K., (2001) Proc.Natl.Acad.Sci.U.S.A.98:2268-2273) and tyrosyl (referring to (for example), Edwards, H. and Schimmel, P. (1990) Mol.Cell.Biol.10:1633-1641) system that obtains of synthetic enzyme has been described and has been applicable to yeast saccharomyces cerevisiae.Intestinal bacteria tyrosyl system has been used for incorporating 3-iodo-L-tyrosine into mammalian cell in vivo.Referring to, Sakamoto, people such as K., (2002) Nucleic Acids Res.30:4692-4699.
The use of O-tRNA/ aminoacyl-tRNA synthetase relates to the selection of the non-naturally encoded amino acid whose specificity codon of coding.Though can use any codon, need usually to select seldom or the codon from be not used in the cell of wherein expressing the O-tRNA/ aminoacyl-tRNA synthetase.For example, exemplary codon comprises nonsense codon, such as terminator codon (amber codon, ocher codon and opal codon), 4 or 4 above base codons and seldom or obsolete other natural 3 base codons.
Known mutation method in the field under can using (include, but is not limited to site specific sudden change, cassette mutagenesis, restriction and select sudden change or the like) selects codon to introduce in the appropriate location in the hGH polynucleotide encoding sequence specificity.
Generation can be described in Wang, people such as L., Science 292:498-500 (2001) in order to incorporate the method for non-naturally encoded amino acid whose component such as the right protein biosynthesizing device of O-RS, O-tRNA and orthogonal O-tRNA/O-RS into; Chin, people such as J.W., J.Am.Chem.Soc.124:9026-9027 (2002); Zhang, people such as Z. are among the Biochemistry 42:6735-6746 (2003).Be used for incorporating into non-naturally encoded amino acid whose method and composition in vivo and be described in the U.S. Patent Application Publication case 2003/0082575 (the 10/126th, No. 927), described patent is to incorporate this paper by reference into.The right method of orthogonal tRNA-tRNA synthetic enzyme that is used to select be applicable to the system that in vivo translates of organism also is described in U.S. Patent Application Publication case 2003/0082575 the (the 10/126th, No. 927) and 2003/0108885 the (the 10/126th, No. 931) in, described patent is to incorporate this paper by reference into.The title of all incorporating this paper by reference into is No. 04/035743, the open case WO of the PCT of " Site Specific Incorporation of Keto Amino Acids into Proteins ", and it is right to describe the orthogonal RS and the tRNA that are used to incorporate into keto amino acid.To describe the orthogonal RS and the tRNA that are used for non-naturally encoded amino acid is incorporated into eukaryotic host cell for WO04/094593 number right for the open case of the PCT of " Expanding the Eukaryotic Genetic Code " for the title of all incorporating this paper by reference into.
The method that is used to produce at least one orthogonal aminoacyl-tRNA synthetase (O-RS) of recombinating comprises: (a) produce (sudden change optionally) the RS storehouse that is obtained by at least one aminoacyl-tRNA synthetase (RS) from first organism, described first organism includes, but is not limited to prokaryotic organism or the eukaryote such as Methanococcus jannaschii, horizontal river virus, Halobacterium, bacillus coli, the ancient green-ball bacterium of flicker, fireball bacterium, pick Yue Shi hot-bulb bacterium, the hot bacterium of the quick gas of thermophile bacteria, height thermophile bacteria (T.thermophilus) or the like; (b) select the member in (and/or screening) RS (Tu Bian RS optionally) storehouse, described member is the orthogonal tRNA of amino acidylate (O-tRNA) in the presence of non-naturally encoded amino acid and natural amino acid, thereby the pond of active (sudden change optionally) RS is provided; And/or, (c) the active RS (including, but is not limited to the RS that suddenlys change) in selection (optionally passing through Negative Selection) pond, there is not preferentially amino acidylate O-tRNA under the non-naturally encoded amino acid whose situation in described active RS, thereby at least one reorganization O-RS is provided; Wherein at least one reorganization O-RS preferentially uses non-naturally encoded amino amino acidylate O-tRNA.
In one embodiment, RS is nonactive RS.Nonactive RS can produce by active RS is suddenlyd change.For example, nonactive RS can by make at least about a kind, at least about 2 kinds, at least about 3 kinds, at least about 4 kinds, at least about 5 kinds, at least about 6 kinds or produce at least about the different amino acid that 1O kind or the amino acid mutation more than 10 kinds become to include, but is not limited to L-Ala.
Known various technology in the affiliated field can be used in the storehouse of sudden change RS, include, but is not limited to the appropriate design based on protein three-dimensional RS structure, or RS Nucleotide at random or the sudden change in the appropriate design technology produce.For example, sudden change RS can by site specific sudden change, random mutation, difference produce recombination mutation, chimeric construct body, appropriate design and describe herein or affiliated field in known additive method produce.
In one embodiment, select the active member in (and/or screening) RS (Tu Bian RS optionally) storehouse, promptly (include, but is not limited to) member of the orthogonal tRNA of amino acidylate (O-tRNA) in the presence of non-naturally encoded amino acid and natural amino acid, comprise: will include, but is not limited to the positivity selection of antibiotics resistance gene or the like or selection markers and (sudden change optionally) RS storehouse is introduced in a plurality of cells, wherein positivity is selected and/or selection markers comprises at least one and includes, but is not limited to amber codon, the selection codon of ocher codon or opal codon; A plurality of cells are grown in the presence of selective agent; Select codon by at least one that suppresses in positivity selection or the selection markers, be identified in selection and/or selective agent and have the cell of (or showing specific reaction) of survival down, thereby the subclass of the cell of being selected by positivity in the pond of containing activity (optionally suddenling change) RS is provided.Optionally, selection and/or selective agent concentration can change.
On the one hand, the positivity selective marker is that the selection codon in E.C. 2.3.1.28 (CAT) gene and the CAT gene is the amber terminator codon.Optionally, the positivity selective marker is that the selection codon in β-Nei Xiananmei and the β-Nei Xiananmei gene is the amber terminator codon.On the other hand, the positivity selection markers comprises fluorescent mark or luminous selection markers or based on the selection markers (including, but is not limited to cell surface marker) of avidity.
In one embodiment, negativity select or the screening pond in the active RS that does not have preferential amino acidylate O-tRNA under the non-naturally encoded amino acid whose situation (sudden change optionally), comprise: negativity selection or selection markers are introduced in a plurality of cells of second organism with the pond with activity (the sudden change optionally) RS from positivity selection or screening, wherein negativity selection or selection markers comprise at least one selection codon (include, but is not limited to antibiotics resistance gene, include, but is not limited to E.C. 2.3.1.28 (CAT) gene); With, be identified in first substratum that replenishes by non-naturally encoded amino acid and screening or selective agent survival or show the specificity screening reaction, but in second substratum that does not replenish, fail to survive or shows the cell of specific reaction, thereby survivaling cell or the cell through screening with at least one reorganization O-RS is provided by non-naturally encoded amino acid and selection or selective agent.For example, the CAT identifying schemes plays positivity selection and/or negativity screening effect in the determining of suitable O-RS recombinant chou.For example, on optionally, have or do not have one or more non-naturally encoded amino acid whose CAT the containing grown cultures dish of (it comprises at least one and selects codon), duplicate clonal pond.Therefore think, contain reorganization O-RS containing the group of growing on the non-naturally encoded amino acid whose culture plate specially.On the one hand, select the concentration of (and/or screening) agent to change.In certain aspects, first organism is different with second organism.Therefore, first organism and/or second organism optionally comprise: prokaryotic organism, eukaryote, Mammals, bacillus coli, fungi, yeast, archeobacteria (archaebacterium), eubacterium, plant, insect, protobiont or the like.In other embodiments, selection markers comprises fluorescent mark or luminous selection markers or based on the selection markers of avidity.
In another embodiment, activity (the sudden change optionally) RS that selects or screen (including, but is not limited to negativity selects) pond comprises: make to have the pond separation of selecting the activity sudden change RS of step (b) from positivity; The pond of negativity selection or selection markers and activity (sudden change optionally) RS is introduced in a plurality of cells of second organism, wherein negativity selection or selection markers comprise at least one selection codon (include, but is not limited to comprise the toxicity marker gene that at least one selects codon, include, but is not limited to rnase barnase gene); With, be identified in not by survival in first substratum of non-naturally encoded amino acid supplementation or the reaction of displaying specificity screening, but in second substratum, fail to survive or show the cell that specificity screening reacts by non-naturally encoded amino acid supplementation, thereby survivaling cell or the cell through screening with at least one reorganization O-RS are provided, and wherein at least one reorganization O-RS is specific concerning non-naturally encoded amino acid.On the one hand, at least one selects codon to comprise about selection codon more than 2 or 2.Described embodiment optionally can comprise, wherein at least one selection codon comprises the selection codon more than 2 or 2, wherein first organism is different (include, but is not limited to, each organism optionally is (including, but is not limited to) prokaryotic organism, eukaryote, Mammals, bacillus coli, fungi, yeast, archeobacteria, eubacterium, plant, insect, protobiont or the like) with second organism.In addition, some aspects comprise that wherein the negativity selective marker comprises rnase barnase gene (it comprises at least one and selects codon).Other aspects comprise, wherein selection markers optionally comprises fluorescent mark or luminous selection markers or based on the selection markers of avidity.Among the embodiment in this article, screening and/or the variation of selecting optionally to comprise screening and/or selecting severity.
In one embodiment, the method for producing at least one orthogonal aminoacyl-tRNA synthetase (O-RS) of recombinating can further comprise: (d) separate at least one reorganization O-RS; (e) produce second group of O-RS that obtains by at least one reorganization O-RS (sudden change optionally); With, (f) repeating step (b) and (c) till the sudden change O-RS of the ability that obtains to comprise preferential amino acidylate O-tRNA.Optionally, repeating step (d)-(f) includes, but is not limited to repetition at least about twice.On the one hand, second group of O-RS by the sudden change that at least one reorganization O-RS obtains can be by including, but is not limited to the sudden change of random mutation, site specific sudden change, and reorganization or its make up and produce.
In aforesaid method, include, but is not limited to the severity of positivity selection/screening step (b), negativity selection/screening step (c) or positivity and negativity selection/screening step (b) and selection (c)/screening step, optionally comprise selection/screening severity is changed.In another embodiment, positivity selection/screening step (b), negativity selection/screening step (c) or positivity and negativity selection/screening step (b) and c) comprise and use the report body report that wherein body is to detect or report that wherein body passes through luminous detection by fluorescent activation cell divide art (FACS).Optionally, the report body is to show on cell surface, phage display device or the like, and is based on the avidity that relates to non-naturally encoded amino acid or analogue or catalytic activity and selects.In one embodiment, the synthetic enzyme of sudden change is to show on cell surface, phage display device or the like.
The method of producing the orthogonal tRNA of reorganization (O-tRNA) comprising: the storehouse that (a) produces the sudden change tRNA that is obtained by the tRNA that includes, but is not limited to suppressor gene tRNA from least one of first organism; (b) select (to include, but is not limited to, negativity is selected) or the screening storehouse under not existing from the situation of the aminoacyl-tRNA synthetase (RS) of first organism by (sudden change optionally) tRNA from the amino acidylate of RS of second organism, thereby tRNA is provided the pond of (sudden change optionally); With, (c) select or screen the member of the amino acidylate of being introduced of orthogonal RS (O-RS) in tRNA (sudden change optionally) pond, thereby at least one reorganization O-tRNA is provided; Wherein at least one reorganization O-tRNA picks out and selects codon and effectively do not picked out by the RS from second organism and by the preferentially amino acidylate of O-RS.In certain embodiments, at least one tRNA is suppressor gene tRNA and/or the 3 base codons that comprise the uniqueness with natural and/or non-natural base, or nonsense codon, rare codon, non-natural codon, the codon that comprises at least 4 bases, amber codon, ocher codon or opal terminator codon.In one embodiment, reorganization O-tRNA has the orthogonality of improvement.Should be appreciated that in certain embodiments, O-tRNA optionally need not to modify and can bring into first organism from second organism.In various embodiments, first organism and second organism are identical or different and optionally are selected from (including, but is not limited to) prokaryotic organism (including, but is not limited to Methanococcus jannaschii, horizontal river virus, bacillus coli, Halobacterium or the like), eukaryote, Mammals, fungi, yeast, archeobacteria, eubacterium, plant, insect, protobiont or the like.In addition, reorganization tRNA optionally comes amino acidylate by non-naturally encoded amino acid, and wherein non-naturally encoded amino acid is in vivo, natively or the biosynthesizing by genetic manipulation.Non-naturally encoded amino acid optionally is added to the growth medium that is used at least the first organism or second organism.
On the one hand, select (to include, but is not limited to, negativity is selected) or the screening storehouse by (sudden change optionally) tRNA (step (b) of the amino acidylate of aminoacyl-tRNA synthetase, comprise: toxicity marker gene and (sudden change optionally) tRNA storehouse are incorporated in a plurality of cells from second organism, and its toxic marker gene comprises at least one and selects codon (or causing the gene of toxigenicity agent or electrostatic agent or wherein said marker gene is comprised at least one organism of selecting codon being necessary gene); With, the cell of selection survival, wherein survivaling cell contains the pond of (the sudden change optionally) tRNA that comprises at least one orthogonal tRNA or non-functional tRNA.For example, survivaling cell can be selected by using the calibrating of cell density comparing rate.
On the other hand, the toxicity marker gene can comprise the selection codon more than 2 or 2.In another embodiment of described method, the toxicity marker gene is a rnase barnase gene, and wherein rnase barnase gene comprises at least one amber codon.Optionally, rnase barnase gene can comprise the amber codon more than 2 or 2.
In one embodiment, select or the member of the amino acidylate of being introduced of orthogonal RS (O-RS) in the pond of screening (sudden change optionally) tRNA can comprise: positivity is selected or the selection markers gene is incorporated in a plurality of cells from second organism together with the pond of O-RS and (sudden change optionally) tRNA, wherein the positivity marker gene comprises drug resistance gene and (includes, but is not limited to the β-Nei Xiananmei gene, it comprises at least one and selects codon, such as at least one amber terminator codon) or be necessary gene to organism, or cause toxic agents toxicide gene; With; be identified in and include, but is not limited to antibiotic selection or the survivaling cell of the following growth of selective agent existence or screened cell; thereby provide the pond of the cell that has at least one reorganization tRNA; wherein respond at least one and select codon, at least one reorganization tRNA by the amino acidylate of O-RS and with aminoacid insertion in translational product by positivity marker gene coding.In another embodiment, the concentration of selection and/or selective agent changes.
The right method of specificity O-tRNA/O-RS that produces is provided.Method comprises: the storehouse that (a) produces the sudden change tRNA that is obtained by at least one tRNA from first organism; (b) negativity select or the screening storehouse under not existing from the situation of the aminoacyl-tRNA synthetase (RS) of first organism by (sudden change optionally) tRNA from the amino acidylate of RS of second organism, thereby the pond of (sudden change optionally) tRNA is provided; The member of the amino acidylate of being introduced of orthogonal RS (O-RS) in the pond of (c) selection or screening (sudden change optionally) tRNA, thus at least one reorganization O-tRNA is provided.At least one reorganization O-tRNA picks out the selection codon and is not effectively picked out by the RS from second organism, and by the preferentially amino acidylate of O-RS.Described method comprises that also (d) produces (sudden change optionally) the RS storehouse that is obtained by at least one aminoacyl-tRNA synthetase (RS) from the 3rd organism; (e) select or screen member of at least one reorganization O-tRNA of preferentially amino acidylate in the presence of non-naturally encoded amino acid and natural amino acid in sudden change RS storehouse, thereby the pond of active (sudden change optionally) RS is provided; With; (f) negativity selection or screening pond does not exist activity (the optionally suddenling change) RS of at least one reorganization O-tRNA of preferentially amino acidylate under the non-naturally encoded amino acid whose situation; thereby provide at least one specificity O-tRNA/O-RS right, wherein at least one specificity O-tRNA/O-RS is specific reorganization O-RS and at least one reorganization O-tRNA to comprising at least one to non-naturally encoded amino acid.Comprise that the specificity O-tRNA/O-RS that is produced by described method is right.For example, specificity O-tRNA/O-RS is right to comprising (including, but is not limited to) mutRNATyr-mutTyrRS, such as mutRNATyr-SS12TyrRS to, mutRNALeu-mutLeuRS to, mutRNAThr-mutThrRS to, mutRNAGlu-mutGluRS to or the like.In addition, described method comprises that wherein first organism is identical (including, but is not limited to Methanococcus jannaschii) with the 3rd organism.
Selection is applicable to that the right method of orthogonal tRNA-tRNA synthetic enzyme of the system that in vivo translates of second organism is also included among the present invention.Described method comprises: marker gene, tRNA and the aminoacyl-tRNA synthetase (RS) that separates from first organism or obtain are incorporated into first group of cell from second organism; Marker gene and tRNA are incorporated in the replicating cell group from second organism; With select first group in the survivaling cell of in the replicating cell group, failing to survive, or the cell of the described reaction of generation of specificity screening reaction is not showed in screening in the replicating cell group, wherein first group and replicating cell group be select or selective agent in the presence of grow, it is right that wherein survivaling cell or the cell that screened comprise the orthogonal tRNA-tRNA synthetic enzyme of the system that in vivo translates that is applicable to second organism.In one embodiment, relatively and select or screening comprises in vivo complementary calibrating.The concentration of selection or selective agent can be variation.
Organism of the present invention comprises various organisms and various combination.For example, first and second organisms of method of the present invention can be identical or different.In one embodiment, organism is prokaryotic organism optionally, includes, but is not limited to Methanococcus jannaschii, horizontal river virus, Halobacterium, bacillus coli, the ancient green-ball bacterium of flicker, fireball bacterium, pick Yue Shi hot-bulb bacterium, the hot bacterium of the quick gas of thermophile bacteria, height thermophile bacteria or the like.Perhaps, organism optionally comprises most eukaryotes, include, but is not limited to plant (including, but is not limited to), algae, protobiont, fungi (including, but is not limited to yeast or the like), animal (including, but is not limited to Mammals, insect, arthropods or the like) or the like such as the complicated plant of monocotyledons or dicotyledons.In another embodiment, second organism is prokaryotic organism, includes, but is not limited to Methanococcus jannaschii, horizontal river virus, Halobacterium, bacillus coli, the ancient green-ball bacterium of flicker, Halobacterium, fireball bacterium, pick Yue Shi hot-bulb bacterium, the hot bacterium of the quick gas of thermophile bacteria, height thermophile bacteria or the like.Perhaps, second organism can be most eukaryotes, includes, but is not limited to yeast, zooblast, vegetable cell, fungi, mammalian cell or the like.In various embodiments, first and second organisms are different.
VII. the location of amino acid in the hGH polypeptide that exist of non-natural
The present invention is contained the amino acid that one or more non-naturals are existed and is incorporated in the GH polypeptide of hGH for example.The amino acid that one or more non-naturals exist can be incorporated in the active concrete position of not destroying polypeptide.Described incorporating into can be by including, but is not limited to replace that hydrophobic amino acid, huge amino acid, the hydrophilic amino acid of huge aminoacid replacement replace hydrophilic amino acid and/or aminoacid insertion that non-natural is existed does not replace and do not realize for " conservative " in the active desired position with hydrophobic amino acid.
For example the zone of the GH of hGH can be described as follows, and wherein the amino acid position among the hGH is instructed in (SEQ ID NO:2) in the middle row:
Spiral A spiral B spiral C spiral D
[1-5]-[6-33]-[34-74]-[75-96]-[97-105]-[106-129]-[130-153]-[154-183]-[184-191]
The terminal A-B ring of N-B-C ring C-D ring C-end
Can use various biological chemistries and structural approach are used in the GH of for example hGH polypeptide selecting, the desired position that replaces with non-naturally encoded amino acid.Any position that it is evident that polypeptide chain concerning the those skilled in the art is suitable for selecting to incorporate into non-naturally encoded amino acid, and selection may be based on appropriate design or by selecting at random to be used for any concrete desired purpose or non-concrete desired purpose.Select desired position can be used to produce GH, for example have any desired character or active hGH molecule, include, but is not limited to agonist, super agonist, anti-agonist, antagonist, receptors bind conditioning agent, receptor activity modulators, being used for dimer or polymer forms, compare with primary molecule and not change activity or character, or handle any polypeptide physics or chemical property such as solubility, aggregation or stability.For example, the location in the needed polypeptide of biological activity of the GH of for example hGH polypeptide can use known point mutation analysis in the affiliated field, L-Ala scanning or homologue scanning method to discern.Referring to (for example), Cunningham, B. and Wells, J., Science, 244:1081-1085 (1989) (discerning 14 to GH, for example the residue of hGH biological activity key) and Cunningham, people Science 243:1330-1336 (1989) such as B. (using homologue scanning sudden change identification antibody and receptor antigen decision base).No. the 5th, 580,723, United States Patent (USP) incorporating this paper by reference into; The 5th, 834, No. 250; The 6th, 013, No. 478; The 6th, 428, No. 954; With the 6th, 451, No. 561 descriptions are by discerning the active active territory that influence has the polypeptide of target substance, the method for the 26S Proteasome Structure and Function of the polypeptide of systems analysis such as hGH.Be different to be identified as and decide on the desired activity of searching polypeptide, and be the good candidate that the non-naturally encoded amino acid of use replaces the residue of the described residue of biological activity key by L-Ala or homologue scanning sudden change.Perhaps, be identified as position, also can decide on the desired activity of searching polypeptide once more, and be the good candidate of using non-naturally encoded amino acid to replace the biological activity key.Another alternative method is simply on each position on the polypeptide chain, replaces continuously and observes effect to polypeptide active with non-naturally encoded amino acid.It will be apparent to those skilled in the art that and be used for selecting non-natural aminoacid replacement is applicable to the present invention to any way, technology or the method for the position of any polypeptide.
Also can study the structure and the activity of the naturally occurring mutant of the hGH polypeptide that contains disappearance, to determine the to allow proteinic zone that replaces with non-naturally encoded amino acid.Concerning hGH, referring to (for example), people such as Kostyo, Biochem.Biophys.Acta, 925:314 (1987); Lewis, people such as U., J.Biol.Chem., 253:2679-2687 (1978).In a similar manner, protease digestion and monoclonal antibody can be undertaken zone in conjunction with the hGH of hGH acceptor in order to identification.Referring to (for example), Cunningham, people Science 243:1330-1336 (1989) such as B.; Mills, people such as J., Endocrinology, 107:391-399 (1980); Li, C., Mol.Cell.Biochem., 46:31-41 (1982) (indication need not to lose activity and can remove amino acid between the residue 134-149).In case residue that may not allow non-naturally encoded amino acid allowable to replace is eliminated, just can come the influence on each rest position of being substituted in of institute's suggestion from the three-dimensional crystalline structure of hGH and its conjugated protein.Concerning hGH, referring to de Vos, people such as A., Science, 255:306-312 (1992); The all crystals structure of hGH can obtain (comprising 3HHR, 1AXI and 1HWG) (PDB in Protein Data Bank (Protein DataBank), can be at World Wide Web, obtain on the rcsb.org), described database is the centralized data base that contains the macromolecular three-dimensional structure data of protein and nucleic acid.If three-dimensional structure data is unavailable, can set up the secondary of research polypeptide and the model of tertiary structure so.Therefore, the those skilled in the art can discern the amino acid position that available non-naturally encoded amino acid replaces easily.
In certain embodiments, for example the GH polypeptide of hGH of the present invention comprises the amino acid that the non-natural in one or more protein zones that are arranged in the spiral that can not destroy polypeptide or β secondary sheet structure exists.
Incorporate non-naturally encoded amino acid whose exemplary residue into and can be the residue that those (include, but is not limited to position I and position II) from possible receptors bind zone and get rid of, can those be the residues that completely or partially are exposed to solvent, the residue that those have and are close to the minimum interaction of hydrogen bond of residue or do not have interaction of hydrogen bond, it can be the residue that those bottom lines are exposed to contiguous reactive residue, and can be those as by in conjunction with or be not joined to the three-dimensional crystalline structure of the hGH of its acceptor, secondary, three grades or quaternary structure are predicted, the residue on highly flexible (including, but is not limited to the C-D ring) or structure in inflexible (the including, but is not limited to the B spiral) zone.
In certain embodiments, one or more non-naturally encoded amino acid be following corresponding to the secondary structure among the hGH more than 1 or 1 with any position in the lower area on incorporate into: corresponding to 1-5 (N-end) from SEQ ID NO:2,6-33 (A spiral), 34-74 (the zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), 97-105 (the zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (zone between C spiral and the D spiral, C-D ring), 154-183 (D spiral), the position of 184-191 (C-end).In other embodiments, for example the GH polypeptide polypeptide of hGH of the present invention comprises the amino acid that at least a amino acid whose non-natural that at least a replacement is arranged at least one zone of the GH of hGH for example exists, and described zone is to be selected from by corresponding to the group that forms with the zone of lower area: the N-end (32-46) that the N-end (1-5) of SEQ ID NO:2, A-B encircle; B-C ring (97-105), C-D encircle (132-149) and C-end (184-191).In certain embodiments, one or more non-naturally encoded amino acid are to incorporate on upper/lower positions at the GH of for example hGH one or more, described position corresponding to: (that is to say before the corresponding amino acid whose position 1 of SEQ ID NO:2 or SEQ ID NO:1 or 3, in the N-end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (that is to say) at proteinic C-terminal place.
Incorporate one or more non-naturally encoded amino acid whose exemplary positions into and comprise position: from the corresponding amino acid whose position 29 of SEQ ID NO:2 or SEQ ID NO:1 or 3 corresponding to following position, 30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 or its any combination.
The subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises the position corresponding to following position: from the corresponding amino acid whose position 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 of SEQ ID NO:2 or SEQ ID NO:1 or 3 and 187 or its any combination.For example Study of Interaction indication of the crystalline structure of the GH of hGH and itself and the GH acceptor of for example hGH, the side chain of described amino-acid residue are that completely or partially that can reach and non-naturally encoded amino acid whose side chain can point in the solvent away from protein surface for solvent.
Incorporate one or more non-naturally encoded amino acid whose example location into and comprise position: from the corresponding amino acid whose position 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 of SEQ ID NO:2 or SEQ ID NO:1 or 3 and 155 or its any combination corresponding to following position.For example the crystalline structure of the GH of hGH and its Study of Interaction with the GH acceptor of for example hGH are indicated, and the side chain of described amino-acid residue is fully to be exposed to side chain solvent and natural residue to point in the solvent.
The subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises the position corresponding to following position: from corresponding amino acid whose position 30,74,103 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.Another subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises the position corresponding to following position: from corresponding amino acid whose 35,92,143,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.Another subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises the position corresponding to following position: from corresponding amino acid whose position 35,92,131,134,143,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.Another subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises the position corresponding to following position: from corresponding amino acid whose position 30,35,74,92,103,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.Another subclass of incorporating one or more non-naturally encoded amino acid whose exemplary positions into comprises the position corresponding to following position: from corresponding amino acid whose position 35,92,143,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.In certain embodiments, incorporating one or more non-naturally encoded amino acid whose positions into comprises corresponding to the position from the corresponding amino acid whose position 35 of SEQ ID NO:2 or SEQ ID NO:1 or 3.
In certain embodiments, at least a non-naturally encoded amino acid of incorporating among the GH of hGH for example contains for example carbonyl of ketone group.In certain embodiments, at least a non-naturally encoded amino acid of incorporating among the GH of hGH for example is to acetylphenylalanine.For example the GH of hGH contains among a plurality of non-naturally encoded more amino acid whose embodiment therein, and more than one the non-naturally encoded amino acid of incorporating among the GH of hGH for example is to acetylphenylalanine.For example the GH of hGH contains among a plurality of non-naturally encoded more amino acid whose embodiment therein, and all non-naturally encoded amino acid substantially of incorporating among the GH of hGH for example are to acetylphenylalanine.
In certain embodiments, the amino acid that non-natural exists is to be connected with water-soluble polymers on including, but is not limited to corresponding to one or more positions with the position of upper/lower positions: (that is to say before the position 1, in the N-end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (that is to say) (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3) at proteinic C-terminal place.In certain embodiments, the amino acid that exists of non-natural is to connect with water-soluble polymers on the position that includes, but is not limited to corresponding to one or more the following stated persons' position: 30,35,74,92,103,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, the amino acid that exists of non-natural is to connect with water-soluble polymers on the position that includes, but is not limited to corresponding to one or more the following stated persons' position: 35,92,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, the amino acid that exists of non-natural is to connect with water-soluble polymers on the position that includes, but is not limited to corresponding to one or more the following stated persons' position: from corresponding amino acid whose position 35,92,131,134,143,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.In certain embodiments, the amino acid that exists of non-natural is to connect with water-soluble polymers on the position that includes, but is not limited to corresponding to one or more the following stated persons' position: from corresponding amino acid whose position 30,35,74,92,103,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.In certain embodiments, the amino acid that exists of non-natural is to connect with water-soluble polymers on the position that includes, but is not limited to corresponding to one or more the following stated persons' position: from corresponding amino acid whose position 35,92,143,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.In certain embodiments, the amino acid of non-natural existence is to be connected with water-soluble polymers on corresponding to (but being not limited to) position from the corresponding amino acid whose position 35 of SEQ ID NO:2 or SEQ ID NO:1 or 3.
In certain embodiments, the water-soluble polymers that is connected with the GH of for example hGH comprises one or more peg molecules (PEG).For example the polymkeric substance of PEG can be straight chain or ramose.Usually, the straight-chain polymer that is used for for example PEG of the present invention can have about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.Usually, the branched polymer that is used for for example PEG of the present invention can have about 1 to about 100kDa, and about 30 to about 50kDa, or the MW of about 40kDa.Polymkeric substance such as PEG is further described in herein.In certain embodiments, for example the key between the water-soluble polymers of the GH of hGH and for example PEG is the oxime key.
Some embodiment of the present invention is contained the composition of the GH that comprises for example hGH that is connected with at least a water-soluble polymers by covalent linkage, and wherein covalent linkage is the oxime key.In certain embodiments, water-soluble polymers is the PEG of straight chain PEG for example.In some embodiment of containing at least a straight chain PEG that is connected with the GH of for example hGH by the oxime key, PEG can have about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.In some embodiment of containing the straight chain PEG that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 30kDa.In some embodiment of containing at least a PEG of branch that is connected with the GH of for example hGH by the oxime key, PEG can have about 0.1 to about 100kDa or about 30 to about 50kDa, or the MW of about 40kDa.In some embodiment of containing the PEG of branch that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 40kDa.In certain embodiments, GH is the GH of for example hGH and in some described embodiment, and for example the GH of hGH has the sequence that is same as SEQ ID NO:2 at least about 80%; In certain embodiments, for example the GH of hGH has sequence for the sequence of SEQ ID NO:2.In certain embodiments, for example the GH of hGH contains at least a non-naturally encoded amino acid; In some described embodiment, at least one oxime key is between non-naturally encoded amino acid and at least a water-soluble polymers.In certain embodiments, non-naturally encoded amino acid contains the carbonyl such as ketone group; In certain embodiments, non-naturally encoded amino acid is to acetylphenylalanine.In certain embodiments, be on position, to be substituted to acetylphenylalanine corresponding to the position 35 of SEQ ID NO:2.
Therefore, in certain embodiments, the invention provides the GH of for example hGH that is connected with the water-soluble polymers of at least a for example PEG by covalent linkage, wherein covalent linkage is the oxime key.In certain embodiments, water-soluble polymers is that PEG and PEG are straight chain PEG.In described embodiment, straight chain PEG has about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.In some embodiment of containing the straight chain PEG that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 30kDa.In certain embodiments, water-soluble polymers is the PEG for the PEG of branch.In described embodiment, the PEG of branch has about 1 to about 100kDa, or about 30 to about 50kDa, or the MW of about 40kDa.In some embodiment of containing the PEG of branch that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 40kDa.
In certain embodiments, the invention provides for example GH of hGH, wherein for example the GH of hGH contains non-naturally encoded amino acid, wherein GH is connected by the water-soluble polymers of covalent linkage with at least a for example PEG, and covalent linkage is the oxime key between the water-soluble polymers of non-natural amino acids coding and for example PEG.In certain embodiments, non-naturally encoded amino acid is to incorporate on the position corresponding to the position 35 of SEQ ID NO:2 among the GH of hGH for example.At water-soluble polymers is among some embodiment of PEG, and PEG is straight chain PEG.In described embodiment, straight chain PEG has about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.In some embodiment of containing the straight chain PEG that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 30kDa.At water-soluble polymers is among some embodiment of PEG, and PEG is the PEG of branch.In described embodiment, the PEG of branch has about 1 to about 100kDa, or about 30 to about 50kDa, or the MW of about 40kDa.In some embodiment of containing the PEG of branch that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 40kDa.
In certain embodiments, the invention provides for example GH of hGH, wherein for example the GH of hGH contains the promising non-naturally encoded amino acid whose non-naturally encoded amino acid that contains carbonyl, GH is connected by the water-soluble polymers of covalent linkage with at least a for example PEG, and covalent linkage is the oxime key between the water-soluble polymers that contains carbonylamino acid and for example PEG of non-natural coding.In certain embodiments, the non-naturally encoded carbonylamino acid that contains is to incorporate on the position corresponding to the position 35 of SEQID NO:2 among the GH of hGH for example.At water-soluble polymers is among some embodiment of PEG, and PEG is straight chain PEG.In described embodiment, straight chain PEG has about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.In some embodiment of containing the straight chain PEG that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 30kDa.At water-soluble polymers is among some embodiment of PEG, and PEG is the PEG of branch.In described embodiment, the PEG of branch has about 1 to about 100kDa, or about 30 to about 50kDa, or the MW of about 40kDa.In some embodiment of containing the PEG of branch that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 40kDa.
In certain embodiments, the invention provides the GH that contains the non-naturally encoded amino acid whose for example hGH that comprises ketone group, wherein GH is connected by the water-soluble polymers of covalent linkage with at least a for example PEG, and covalent linkage is the oxime key that contains between the water-soluble polymers of the non-naturally encoded amino acid of ketone group and for example PEG.In certain embodiments, the non-naturally encoded amino acid that contains ketone group is to incorporate on the position corresponding to the position 35 of SEQ ID NO:2 among the GH of hGH for example.At water-soluble polymers is among some embodiment of PEG, and PEG is straight chain PEG.In described embodiment, straight chain PEG has about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.In some embodiment of containing the straight chain PEG that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 30kDa.At water-soluble polymers is among some embodiment of PEG, and PEG is the PEG of branch.In described embodiment, the PEG of branch has about 1 to about 100kDa, or about 30 to about 50kDa, or the MW of about 40kDa.In some embodiment of containing the PEG of branch that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 40kDa.
In certain embodiments, the invention provides the GH that contains promising non-naturally encoded amino acid whose for example hGH to acetylphenylalanine, wherein GH is connected by the water-soluble polymers of covalent linkage with at least a for example PEG, and covalent linkage is to the oxime key between the water-soluble polymers of acetylphenylalanine and for example PEG.In certain embodiments, be on position, to incorporate among the GH of hGH for example to acetylphenylalanine corresponding to the position 35 of SEQ ID NO:2.At water-soluble polymers is among some embodiment of PEG, and PEG is straight chain PEG.In described embodiment, straight chain PEG has about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.In some embodiment of containing the straight chain PEG that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 30kDa.At water-soluble polymers is among some embodiment of PEG, and PEG is the PEG of branch.In described embodiment, the PEG of branch has about 1 to about 100kDa, or about 30 to about 50kDa, or the MW of about 40kDa.In some embodiment of containing the PEG of branch that is connected with the GH of for example hGH by the oxime key, PEG has the MW of about 40kDa.
In certain embodiments, the invention provides for example GH of hGH, it comprise SEQ ID NO:2 and for example the GH of hGH be on corresponding to the position of the position 35 of SEQ ID NO:2 through acetylphenylalanine is replaced, described acetylphenylalanine is connected to the straight chain PEG of the MW with about 30kDa by the oxime key.
In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and is including, but is not limited to corresponding to substituted at least a non-naturally encoded amino acid on one or more positions of the position of upper/lower positions: (that is to say before the position 1, in the N-end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (that is to say) (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3) at proteinic C-terminal place.In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and including, but is not limited to corresponding to substituted at least a non-naturally encoded amino acid on one or more positions of the position of upper/lower positions: 30,35,74,92,103,143,145 (the corresponding amino acid of SEQID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and including, but is not limited to corresponding to substituted at least a non-naturally encoded amino acid on one or more positions of the position of upper/lower positions: 35,92,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and including, but is not limited to corresponding to substituted at least a non-naturally encoded amino acid on one or more positions of the position of upper/lower positions: from corresponding amino acid whose position 35,92,131,134,143,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and including, but is not limited to corresponding to substituted at least a non-naturally encoded amino acid on one or more positions of the position of upper/lower positions: from corresponding amino acid whose position 30,35,74,92,103,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and including, but is not limited to corresponding to substituted at least a non-naturally encoded amino acid on one or more positions of the position of upper/lower positions: from corresponding amino acid whose position 35,92,143,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO 2, and for example the GH of hGH contains and including, but is not limited to corresponding to substituted at least a non-naturally encoded amino acid on from one or more positions of the position of the corresponding amino acid whose position 35 of SEQ ID NO:2 or SEQ ID NO:1 or 3.PEG is among the embodiment of straight chain PEG therein, and PEG can have about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.
In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and substitutedly on including, but is not limited to corresponding to one or more positions with the position of upper/lower positions is at least a non-naturally encoded amino acid to acetylphenylalanine: (that is to say before the position 1, in the N-end), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (that is to say) (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3) at proteinic C-terminal place.In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and substitutedly on including, but is not limited to corresponding to one or more positions with the position of upper/lower positions is at least a non-naturally encoded amino acid to acetylphenylalanine: 30,35,74,92,103,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and substitutedly on including, but is not limited to corresponding to one or more positions with the position of upper/lower positions is at least a non-naturally encoded amino acid to acetylphenylalanine: 35,92,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and substitutedly on including, but is not limited to corresponding to one or more positions with the position of upper/lower positions is at least a non-naturally encoded amino acid to acetylphenylalanine: from the corresponding amino acid whose position 35 of SEQ ID NO:2 or SEQ ID NO:1 or 3,92,131,134,143,145 or its any combination.In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and substitutedly on including, but is not limited to corresponding to one or more positions with the position of upper/lower positions is at least a non-naturally encoded amino acid to acetylphenylalanine: from the corresponding amino acid whose of SEQ ID NO:2 or SEQ ID NO:1 or 3 is 30,35,74,92,103,145 or its any combination.In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO:2, and for example the GH of hGH contains and substitutedly on including, but is not limited to corresponding to one or more positions with the position of upper/lower positions is at least a non-naturally encoded amino acid to acetylphenylalanine: from the corresponding amino acid whose of SEQ ID NO:2 or SEQ ID NO:1 or 3 is 35,92,143,145 or its any combination.In certain embodiments, the invention provides hormonal composition, it comprises the GH of for example hGH that is connected to the PEG of at least a for example straight chain PEG by the oxime key, wherein for example the GH of hGH comprises the aminoacid sequence of SEQ ID NO 2, and for example the GH of hGH contains and substitutedly on including, but is not limited to corresponding to one or more positions from the position of the corresponding amino acid whose position 35 of SEQ ID NO:2 or SEQ ID NO:1 or 3 is at least a non-naturally encoded amino acid to acetylphenylalanine.PEG is among the embodiment of straight chain PEG therein, and PEG can have about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.
In certain embodiments, the invention provides for example GH of hGH, wherein for example the GH of hGH contains at least a non-naturally encoded amino acid, GH is connected with a plurality of water-soluble polymerss of for example a plurality of PEG by covalent linkage, and one of them or above covalent linkage are the oxime keys between the water-soluble polymers of at least a non-naturally encoded amino acid and for example PEG.For example the GH of hGH can be connected in about 2-100 for example water-soluble polymers of PEG, or the individual for example water-soluble polymers of PEG of about 2-50, or the individual for example water-soluble polymers of PEG of about 2-25, or the individual for example water-soluble polymers of PEG of about 2-10, or the individual for example water-soluble polymers of PEG of about 2-5, or the individual for example water-soluble polymers of PEG of about 5-100, or the individual for example water-soluble polymers of PEG of about 5-50, or the individual for example water-soluble polymers of PEG of about 5-25, or the individual for example water-soluble polymers of PEG of about 5-10, or the individual for example water-soluble polymers of PEG of about 10-100, or the individual for example water-soluble polymers of PEG of about 10-50, or the individual for example water-soluble polymers of PEG of about 10-20, or the individual for example water-soluble polymers of PEG of about 20-100, or the individual for example water-soluble polymers of PEG of about 20-50, or the individual for example water-soluble polymers of PEG of about 50-100.Incorporate on any position that one or more non-naturally encoded amino acid can be described in this article among the GH of hGH for example.In certain embodiments, at least a non-naturally encoded amino acid is to incorporate on the position corresponding to the position 35 of SEQ ID NO:2 among the GH of hGH for example.In certain embodiments, non-naturally encoded amino acid is included as the non-naturally encoded amino acid that contains carbonyl, for example contains the non-naturally encoded amino acid of ketone, such as at least a non-naturally encoded amino acid to acetylphenylalanine.In certain embodiments, for example the GH of hGH comprises acetylphenylalanine.In certain embodiments, be on position, to incorporate among the GH of hGH for example to acetylphenylalanine corresponding to the position 35 of SEQ ID NO:2, be that one of polymkeric substance by oxime key and for example one of PEG is connected wherein to acetylphenylalanine.In certain embodiments, the water-soluble polymers of at least a for example PEG is by being connected with the GH of for example hGH with at least a non-naturally encoded amino acid whose covalent linkage.In certain embodiments, covalent linkage is the oxime key.In certain embodiments, the water-soluble polymers of a plurality of for example PEG is by being connected with the GH of for example hGH with a plurality of non-naturally encoded amino acid whose covalent linkage.In certain embodiments, at least one covalent linkage oxime key; In certain embodiments, a plurality of covalent linkage are oxime keys; In certain embodiments, all substantially keys are oxime keys.The water-soluble polymers of a plurality of for example PEG can be straight chain, ramose or its any combination.In the embodiment that incorporates one or more straight chains PEG into, straight chain PEG has about 0.1 to about 100kDa, or about 1 to about 60kDa, or about 20 to about 40kDa, or the MW of about 30kDa.In the embodiment that incorporates the PEG of one or more branches into, the PEG of branch has about 1 to about 100kDa, or about 30 to about 50kDa, or the MW of about 40kDa.Should be appreciated that, use the embodiment of the water-soluble polymers of a plurality of for example PEG generally speaking will use the described polymkeric substance that compares lower MW among the embodiment that uses single PEG therein.Therefore, in certain embodiments, total MW of a plurality of PEG is about 0.1-500kDa, or about 0.1-200kDa, or about 0.1-100kDa, or about 1-1000kDa, or about 1-500kDa, or about 1-200kDa, or about 1-100kDa, or about 10-1000kDa, or about 10-500kDa, or about 10-200kDa, or about 10-100kDa, or about 10-50kDa, or about 20-1000kDa, or about 20-500kDa, or about 20-200kDa, or about 20-100kDa, or about 20-80kDa, about 20-60kDa, about 5-100kDa, about 5-50kDa, or about 5-20kDa.
Human GH antagonist includes, but is not limited to, on position 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 and 127 or 1 (that is to say) or its any combination in the position in addition in the N-end (SEQ ID NO:2 or SEQ ID NO:1,3 or any other GH sequence in corresponding amino acid) the position on have the described antagonist of replacement.
Can with multiple non-naturally encoded aminoacid replacement or incorporate into hGH for example the GH polypeptide in given position.Generally speaking, select concrete non-naturally encoded amino acid to be used for merging based on research and its acceptor of the three-dimensional crystalline structure of the GH polypeptide of for example hGH, preferred conservative the replacement, (that is to say, non-naturally encoded amino acid based on aryl, such as acetylphenylalanine or O-propargyl tyrosine are replaced Phe, Tyr or Trp) and one of ordinary skill in the art wish to introduce in the GH polypeptide of hGH for example specificity in conjunction with chemical action (for example, if one of ordinary skill in the art want to realize and have the Huisgen[3+2 of the water-soluble polymers of alkynyl moiety] cycloaddition or with the amido linkage formation effect of the water-soluble polymers of the aryl ester that has and incorporate into the phosphine part, so just introduce 4-triazobenzene L-Ala).
In one embodiment, method comprises in addition to be incorporated alpha-non-natural amino acid in the protein into, and wherein alpha-non-natural amino acid comprises first reactive group; With make protein and the molecule that comprises second reactive group (include, but is not limited to mark, dyestuff, polymkeric substance, water-soluble polymers, the derivative of polyoxyethylene glycol, the photocrosslinking agent, radionuclide, cytotoxin compounds, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotides, carbohydrate, water-soluble branch-shape polymer, cyclodextrin, inhibition Yeast Nucleic Acid, biomaterial, nanoparticle, spin label, fluorophore, metallic part, radioactive segment, novel functional group, covalently or non-covalentlyly with the group of other interactions of molecules, the photic part of shrouding, but the part that actinic radiation excites, the intramolecular photosensitization part, vitamin H, the derivative of vitamin H, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, the side chain of elongation, the sugar that carbon connects, the redoxomorphism promoting agent, amino thioic acid sulfoacid, deleterious part, isotope-labeled part, the biophysics probe, phosphorescent group, chemiluminescent group, the close group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable label, small molecules, quantum dot, the nanometer transmitter, the radioactive nuleus thuja acid, the radioactivity transmitter, neutron capture agent, or any combination of above-mentioned substance or any other required compound or material) contact.First reactive group and second reaction-ity group reaction are to be connected to alpha-non-natural amino acid by [3+2] cycloaddition with molecule.In one embodiment, first reactive group is that alkynyl or the azido-part and second reactive group are azido-or alkynyl part.For example, first reactive group is that the alkynyl part (include, but is not limited to alpha-non-natural amino acid to the propargyloxy phenylalanine in) and second reactive group are the azido-parts.In another example, first reactive group is that the azido-part (include, but is not limited to alpha-non-natural amino acid to azido--L-phenylalanine in) and second reactive group are the alkynyl parts.
In some cases, non-naturally encoded aminoacid replacement with the GH polypeptide of for example hGH in other add, replace or the disappearance combination to influence for example other biological feature of the GH polypeptide of hGH.In some cases, other add, replace or disappearance can increase the stability of the GH polypeptide of hGH (including, but is not limited to the resistivity to proteolytic degradation) for example or increase the avidity of the GH polypeptide of hGH for example to its acceptor.In certain embodiments, for example the GH polypeptide of hGH comprises the aminoacid replacement that is selected from the group that is made up of following replacement: F10A, F10H, F10I among the SEQ ID NO:2; M14W, M14Q, M14G; H18D; H21N; G120A; R167N; D171S; E174S; F176Y, I179T or its any combination.In some cases, other add, replace or lack can increase for example solubility of the GH polypeptide of hGH (including, but is not limited to when expressing) in intestinal bacteria or other host cells.In certain embodiments, add, replace or lack and to be increased in the polypeptide solubility after the expression in intestinal bacteria or other recombinant host cells.In certain embodiments, except that being used to incorporate into non-natural amino acid whose position, selection is used for other positions of being replaced by amino acid natural coding or non-natural, to be increased in the polypeptide solubility after expressing in intestinal bacteria or other recombinant host cells.In certain embodiments, for example the GH polypeptide of hGH comprises another interpolation, replacement or disappearance, it regulates the avidity to the GH polypeptide receptor of for example hGH, regulate (including, but is not limited to increases or reduce) receptor dimerization effect, stablize the receptor dimerization thing, regulate circulating half-life, adjustment release or biological usability, promote purifying, or improvement or change concrete dispensing route.For example, except that one or more non-naturally encoded amino acid of introducing as indicated above, introduce one or more following avidity that replaces the GH variant that increases hGH for example to its acceptor: F10A, F10H or F10I; M14W, M14Q or M14G; H18D; H21N; R167N:D171S; E174S; F176Y and I179T.Similarly, for example the GH polypeptide of hGH can comprise chemistry or enzymatic lysis sequence, protease cracking sequence, reactive group, antibody binding domain (including, but is not limited to FLAG or poly-His) or other sequences based on avidity (including, but is not limited to FLAG, poly-His, GST etc.) or link molecule (including, but is not limited to vitamin H), and it improves other features that detect (including, but is not limited to GFP), purifying, pass through transportation, prodrug release or activation, hGH size reduction or the polypeptide of tissue or cytolemma.
In certain embodiments, non-naturally encoded amino acid whose replacement produces for example GH antagonist of hGH.The subclass that is used to incorporate into one or more non-naturally encoded amino acid whose exemplary positions comprises: position 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123,127 or in addition before the position 1 (SEQ ID NO:2 or SEQ ID NO:1,3 corresponding amino acid, or any other GH sequence).In certain embodiments, for example the GH antagonist of hGH comprise make GH serve as antagonist with at least once replacing in the lower area: regional 1-5 (N-end), 6-33 (A spiral), the 34-74 (zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), the 97-105 (zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (zone between C spiral and the D spiral, C-D ring), 154-183 (D spiral), 184-191 (C-end).In other embodiments, incorporate residue in the amino terminal region that non-naturally encoded amino acid whose exemplary position comprises spiral A and a part of spiral C into.In another embodiment, by such as non-naturally encoded aminoacid replacement G120 to azido--L-phenylalanine or O-propargyl-L-tyrosine.In other embodiments, above listed replacement is that the GH polypeptide of hGH becomes for example other replacement combinations of the GH antagonist of hGH with for example making.For example, non-naturally encoded amino acid is that replacement (for example G120R, G120K, G120W, G120Y, G120F or G120E) is located to replace and introduced at G120 place simultaneously in one of position of discerning in this article.In certain embodiments, for example the GH antagonist of hGH comprises the non-naturally encoded amino acid that is connected with water-soluble polymers in the receptors bind zone that is present in the GH molecule of hGH for example.
In some cases, 1,2,3,4,5,6,7,8,9, the amino acid more than 10 kind or 10 kinds is through one or more non-naturally encoded aminoacid replacement.In some cases, for example the GH polypeptide of hGH comprises that in addition one or more non-naturally encoded amino acid are to naturally occurring amino acid whose replacement more than 1,2,3,4,5,6,7,8,9,10 time or 10 times.For example, in certain embodiments, for example the GH of hGH with one or more residues in the lower area through one or more non-naturally encoded aminoacid replacement: 1-5 (N-end); 32-46 (the N-end of A-B ring); 97-105 (B-C ring); And 132-149 (C-D ring); And 184-191 (C-end).In certain embodiments, for example the GH of hGH with one or more residues in the lower area through one or more non-naturally encoded aminoacid replacement: 1-5 (N-end), 6-33 (A spiral), the 34-74 (zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), the 97-105 (zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (zone between C spiral and the D spiral, C-D ring), 154-183 (D spiral), 184-191 (C-end).In some cases, one or more non-naturally encoded residues are to be connected with one or more low molecular weight linears or the PEG of branch (the about 5-20kDa of quality or littler), and then material single with respect to being connected in, high molecular weight PEGs, strengthen binding affinity and suitable serum half-life.
In certain embodiments, for example maximum 2 residues of the following residue of the GH of hGH on upper/lower positions through one or more non-naturally encoded aminoacid replacement: 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187.In some cases, carry out any replacement in the following paired replacement: K38X *And K140X *K41X *And K145X *Y35X *And E88X *Y35X *And F92X *Y35X *And Y143X *F92X *And Y143X *, X wherein *Represent non-naturally encoded amino acid.The non-naturally encoded amino acid whose preferred site that is used to incorporate into two or more comprises the combination of following residue: 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187.The non-naturally encoded amino acid whose especially preferred site that is used to incorporate into two or more comprises the combination of following residue: 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155.
Be used for incorporating two or more non-naturally encoded amino acid into combination that the preferred site of the GH of hGH for example comprises following residue: (that is to say before the position 1 from SEQ ID NO:2, in the N-end), position 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (that is to say) or its any combinations at proteinic C-terminal place.
VIII. measuring h GH polypeptide active and hGH polypeptide are to the avidity of hGH polypeptide receptor
The activity of hGH can be used and include, but is not limited to cell and measure in conjunction with calibrating or to any technology of known some technology in the affiliated field of the pSTAT5 of IM9 cell calibrating.For assessing the biological activity of modified hGH polypeptide, can use the interactional calibrating between monitoring hGH and its acceptor.For example, can use that (ATCC, Manassas measure signal transducer and the activator transcribe the family member, i.e. the calibrating of the tyrosine phosphorylation effect of STAT5 in VA) in human IM-9 lymphocyte clone.Referring to (for example), people such as Silva, Mol.Endocrinol. (1996) 10 (5): 508-518.Make IM-9 cell hunger in calibrating substratum (charcoal of phenol-red free RPMI, 10mM Hepes, 1% heated and inactivated/through FBS, Sodium.alpha.-ketopropionate, penicillin (penicillin) and Streptomycin sulphate (streptomycin) that dextran is handled) overnight, afterwards under 37 ℃, with the hGH polypeptide stimulation 10min of 12-dose point scope.Be excited cell with 1% formaldehyde fixed, use 90% ice cold methanol afterwards permeability on ice 1 hour.The level of STAT5 phosphorylation is by at room temperature, and (MA) cell inner dyeing 30min then detects with the dyeing of PE bonded secondary antibody for Cell Signaling Technology, Beverly with elementary phosphoric acid STAT5 antibody.Sample collecting is to carry out on FACS Array, and (Tree Star Inc., Ashland OR) go up the data that analysis is obtained at Flowio software.The EC50 value is by utilizing SigmaPlot, with average fluorescent strength (MFI) relatively the dose response curve drawn of protein concn obtain.
Perhaps, use the proliferation research of BrdU can be such as in the clone of the BAF3 of rat growth hormone acceptor stable transfection, carrying out.Express BAF3 clone, the clear rat growth hormone acceptor of the ischemic of 2E2-2B12-F4, promptly GHR (L43R) is with 5 * 10 4The density of individual cells/well is applied in the 96 hole culture plates.With the hGH protein activation cell of 12-dose point scope and use 50uM BrdU (Sigma, St.Louis, MO) mark simultaneously.In culture, after 48 hours, at room temperature, fix/permeability cell 30min with the BD cytofix/cytoperm solution (BD Biosciences) of 100ul.For exposing the fixed base of BrdU Kang Yuan Decision, under 37 ℃, with the DNase (Sigma) in 30 micrograms/hole handle through fix/cell of permeability lasts 1 hour.Use the anti-BrdU antibody of APC bonded (BD Biosciences) to carry out fluorescence immunization coloration and make it possible on FACS Array, carry out sample analysis.
The hGH acceptor can be by people such as McFarland, Science, and 245:494-499 (1989) and Leung, people such as D., Nature, 330:537-543 prepares described in (1987).The hGH polypeptide active can use standard or in vitro known or in vivo calibrating mensuration.For example, proliferating cells system (for example expressing the clone of hGH acceptor or lactagogue acceptor) can be in order to monitoring hGH receptors bind in the presence of hGH.Referring to (for example), Clark, people such as R., J.Biol.Chem.271 (36): 21969 (1996); People such as Wada, Mol.Endocrinol.12:146-156 (1998); Gout, people Cancer Res.40 such as P.W., 2433-2436 (1980); WO 99/03887.Concerning the hGH polypeptide that comprises non-natural amino acid whose non-Pegylation or Pegylation, hormone can be by using BIAcore to the avidity of its acceptor TMBiosensor (GEHealthcare) is measured.Referring to (for example), the 5th, 849, No. 535 United States Patent (USP)s; Spencer, people such as S.A., J.Biol.Chem., 263:7862-7867 (1988).Be used to test the active in vivo animal model of hGH and comprise people such as being described in (for example) Clark, the described model among J.Biol.Chem.271 (36): the 21969-21977 (1996).The calibrating that comprises the dimerization ability of one or more non-naturally encoded amino acid whose hGH polypeptide can be by Cunningham, people such as B., Science, 254:821-825 (1991) and Fuh, G. wait the people, Science, 256:1677-1680 carries out described in (1992).The reference of all references and patent are to incorporate this paper by reference into.Above-mentioned writing of reference to calibration method is incomplete, and those skilled in the art will realize that and be applicable to other calibratings that desired end results is tested.
Incorporate into by reference this paper apply on January 28th, 2005 and title describe in detail in addition for No. 2005/0170404 for the U.S. Patent Publication case of " Modified GrowthHormone Polypeptides and Their Uses " amino acid that is used to incorporate into one or more non-naturals and exists, non-naturally encoded amino acid, orthogonal tRNA, orthogonal aminoacyl tRNA synthetase hGH residue and characterize the method for hGH.
IX. measure effectiveness, functional in vivo transformation period and pharmacokinetic parameter
An importance of the present invention is the biological half-life that prolongs, and it is to obtain by the hGH polypeptide of constructing the keying action that has or do not have polypeptide and water-soluble polymers part.The quick reduction of hGH polypeptide serum-concentration has made assessment become important to the biological respinse for the treatment of with bonded and uncombined hGH polypeptide and its variant.Combination of the present invention and uncombined hGH polypeptide and its variant also can have the serum half-life of prolongation after subcutaneous administration or intravenously dispensing, making to measure surely by (for example) ELISA method or by the primary screen Selected Inspection becomes possibility.Can use from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, ELISA TX) or RIA test kit.In vivo the measurement of biological half-life is by execution described herein.
The effectiveness and the functional in vivo transformation period that comprise non-naturally encoded amino acid whose hGH polypeptide can be according to Clark, people such as R., the scheme described in J.Biol.Chem.271 (36): the 21969-21977 (1996) and measuring.
The pharmacokinetic parameter that comprises non-naturally encoded amino acid whose hGH polypeptide can assessment (each treatment group N=5 animal) in normal Sprague-Dawley male rat.Animal will be accepted the 25 micrograms/rat (intravenously) or the 50 micrograms/rat (subcutaneous) of single dose, and will extract about 5-7 blood sample according to predefined time-histories, usually, non-naturally encoded amino acid whose not with water-soluble polymers bonded hGH polypeptide concerning comprising, time-histories contains about 6 hours, and concerning comprising non-naturally encoded amino acid and with water-soluble polymers bonded hGH polypeptide, time-histories contains about 4 hours.In some species, fully studied the pharmacokinetic data of hGH polypeptide, and described data can be directly with by comprising data that non-naturally encoded amino acid whose hGH polypeptide obtained relatively.Referring to people such as Mordenti J., Pharm.Res.8 (11): 1351-59 (1991) is to the research of relevant hGH.
Pharmacokinetic parameter also can be assessed in the primate of for example macaque.Usually, subcutaneous throwing with or intravenously throw and single injection liquid, and monitor serum hGH level in time.
Specific activity according to hGH polypeptide of the present invention can be measured by known various calibratings in the affiliated field.The hGH polypeptide mutein of acquisition and purifying or its segmental biological activity can be tested by the method describing herein or mention or for the method known to the those skilled in the art according to the present invention.
The therapeutic use of X.hGH polypeptide
HGH agonist polypeptide goes for (for example) treatment growth deficiency, immune disorders and is applicable to the promotion heart function.Have the insufficient individuality of growth and comprise (for example), individuality with Te Nashi (Turner) syndromes, the insufficient individuality of GH (comprising children), the about 2-3 of experience before its growth plate sealing, children's (being sometimes referred to as " short and small normal child ") that its normal growth curve slows down or postpones, wherein GH insulin like growth factor-1 (IGF-I) is reacted by the individual of chemical block (that is to say, because of glucocorticoid treatment) or such as the individuality in the adult patients that reduces naturally in the IGF-I reaction to GH.HGH polypeptide of the present invention goes for treating the individuality with following symptom: the paediatrics growth hormone deficiency, special send out property of short and small stature, the Childhood outbreak grow up growth hormone deficiency, the outbreak of growing up grow up growth hormone deficiency or diauxic growth hormone deficiency.The grownup who is had the Adulthood growth hormone deficiency by diagnosis may have pituitary tumor or radiation.The symptom that includes, but is not limited to metabolic syndrome, craniocerebral injury, obesity, osteoporosis or dysthymia disorders may cause adult similar growth hormone deficiency syndromes.
Agonist hGH variant can work to stimulate mammiferous immunity system by increasing mammiferous immunologic function, no matter increase is because antibody-mediated effect or cell-mediated effect, and no matter immunity system is for endogenic to the host with the treatment of hGH polypeptide, still is transplanted to the host recipient (as bone marrow graft) who accepts the hGH polypeptide from donor." immune disorders " comprises any symptom, wherein Ge Ti immunity system have than normal immunity system reduce to antigenic antibody or cell response, described individuality comprises because medicine (for example chemotherapy), and treatment has the described individuality of the little spleen of reduction immunity.Example individuality with immune disorders comprises (for example), the individuality of gerontal patient, experience chemotherapy or radiation-therapy, the individuality, the individuality that recover maybe will to undergo surgery from major disease, (for example have such as hypogammaglobulinaemia deficiency, the gamma-globulin insufficiency of blood that generally changes and selectivity immunoglobulin (Ig) deficiency with AIDS, the IgA deficiency) the congenital and insufficient patient of the B cell day after tomorrow, infection is such as rabic virus, and latent period the patient shorter than patient's immune response; With the individuality that has such as the hereditary illness of diGeorge syndromes.
HGH antagonist polypeptide goes for treating gigantosoma and the reactive malignant tumour of acromegaly, diabetes and the complication (diabetic retinopathy, diabetic neuropathy) that is produced by diabetes, eyes vascular disease (for example, comprising the hyperplasia neovascularization), ephrosis and GH.The eyes vascular disease comprise (for example) retinopathy (being caused by (for example) anemia of prematurity disease or sickle cell disease) and macular degeneration.The reactive malignant tumour of GH comprises (for example), Weir Mu Shi tumour (Wilm ' s tumor), sarcoma (for example, osteogenic sarcoma), breast cancer, colorectal carcinoma, prostate cancer and thyroid carcinoma, with the cancer of the tissue of expressing the GH receptor mrna (that is to say, the cancer of placenta, thymus gland, brain, sialisterium, prostate gland, marrow, skeletal muscle, tracheae, spinal cord, retina, lymphoglandula and from burkitt's lymphoma (Burkitt ' s lymphoma), colorectal carcinoma knurl, lung cancer tumor, lymphoblastic leukemia and melanomatous cancer).
The GH agonist polypeptide of for example hGH of the present invention go for (for example) treatment chronic renal failure, the poor growth relevant with chronic renal insufficiency (CRI), with take off receive relevant of short and small stature, the paediatrics Pa Deweili syndromes (Prader-Willi Syndrome) of syndrome (Turner Syndrome) (PWS), have become thin or the HIV patient of cachexia, less than children (SGA), obesity and the osteoporosis of being born pregnant age.
The hGH polypeptide of the present invention that comprises the hGH of Pegylation, can by any habitual route that includes, but is not limited to parenteral route that is suitable for protein or peptide throw with, for example injection by including, but is not limited to subcutaneous or intravenous injection or any other form or transfusion throw with.Peptide composition can by include, but is not limited to per os, intravenously, peritonaeum Inner, intramuscular, through skin, subcutaneous, local, hypogloeeis or rectal a large amount of routes throw with.The composition that comprises modified or not modified non-natural amino acid polypeptide also can by liposome throw with.Described dispensing route and proper formulation are generally known to the those skilled in the art.Comprise that non-natural amino acid whose hGH polypeptide can use separately or be used in combination with other components that are fit to such as medical supporting agent comprising of Pegylation hGH.
The mean vol of hGH can change and specifically, should be based on titular doctor's recommendation and prescription.The accurate amount of hGH is preferentially considered the following stated factor: the definite type of the symptom of being treated, the patient's who is treated symptom and other compositions in the composition.Amount to be given can be determined based on the therapy of using hGH easily by the those skilled in the art.
Medical composition of the present invention can ways customary be made.
XI. the general recombinant nucleic acid method of using for the present invention
In many embodiment of the present invention, recombination method separates with using, clone and change the nucleic acid of the hGH polypeptide that coding paid close attention to usually.Described embodiment is applicable to (including, but is not limited to) protein expression or uses during the generation of variant, derivative, expressed sequence box or other sequences of being obtained by the hGH polypeptide.In certain embodiments, the encode sequence of polypeptide of the present invention is operationally to be connected with allogeneic promoter.The separation of hGH in host cell and the generation of GH are described in (for example) United States Patent (USP) the 4th, 601,980,4,604,359,4,634,677,4,658,021,4,898,830,5,424,199,5,795,745,5,854,026,5,849,535,6,004,931,6,022,711,6,143,523 and 6,608, in No. 183, described patent is to incorporate this paper by reference into.
The nucleotide sequence that coding comprises non-naturally encoded amino acid whose hGH polypeptide can and then change nucleotide sequence so that the introducing of the Amino acid residue of realizing being correlated with (that is to say based on the aminoacid sequence of parent polypeptide, incorporate into or replace) or remove (that is to say, disappearance or replace) and synthesize.Nucleotide sequence can be expediently by according to the site-directed mutagenesis of conventional process and modified.Perhaps, nucleotide sequence can prepare by chemosynthesis, include, but is not limited to by using the preparation of oligonucleotide synthesizer, wherein oligonucleotide is based on desired amino acid sequence of polypeptide and preferentially is chosen in described codon favourable in the host cell that wherein produces recombinant polypeptide and designs.For example, the small oligonucleotide of the part of the desired polypeptide of some codings can pass through PCR, connection or the synthetic and assembling of connection chain reaction.Referring to (for example), people such as Barany, Proc.Natl.Acad.Sci.88:189-193 (1991); United States Patent (USP) the 6th, 521, No. 427, it is to incorporate this paper by reference into.
The present invention utilizes the routine techniques in the genetic recombination field.The basic article that announcement is used for universal method of the present invention comprises people such as Sambrook, Molecular Cloning, A Laboratory Manual (third edition 2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With Current Protocols inMolecular Biology (people such as Ausubel compiles 1994)).
The general article of describing molecular biotechnology comprises Berger and Kimmel, Guide to Molecular Cloning Technioues, Methods in Enzvmology the 152nd volumeAcademic Press, Inc., San Diego, CA (Berger); People such as Sambrook, Molecular Cloning-A Laboratory Manual (second edition), the 1-3 volume, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (" Sambrook ") and Current Protocols in Molecular Biology.F.M.Ausubel wait the people to compile Current Protocols, a jointventure between Greene Publishing Associates, Inc.and John Wiley ﹠amp; Sons, Inc., (replenishing) (" Ausubel ") by 1999).Sudden change described in described article, the purposes of carrier, promotor and many other related subjects about the generation of (including, but is not limited to) gene or polynucleotide, described gene or polynucleotide comprise being used to produce and comprise alpha-non-natural amino acid, quadrature tRNA, quadrature tRNA synthetic enzyme and its right proteinic selection codon.
The present invention uses various types of sudden changes to be used for various purposes, include, but is not limited to produce novel synthetic enzyme or tRNA, make the sudden change of tRNA molecule, make the polynucleotide sudden change of coding synthetic enzyme, produce the storehouse of tRNA, produce the storehouse of synthetic enzyme, produce and select codon, the selection codon of coding alpha-non-natural amino acid is inserted in the protein of being paid close attention to or polypeptide.Described sudden change includes, but is not limited to location, random point mutation, homologous recombination, DNA slippage or other return mutation method, chimeric construction, use contains sudden change, the oligonucleotide positional mutation of the uridylic of template, the dna mutation of modifying through thiophosphatephosphorothioate, the sudden change of using gapped duplex DNA or the like, or its any combination.Other appropriate methodologies comprise a mispairing reparation, use to repair the sudden change of disappearance host strain, restriction selects and restriction-purifying, deletion mutantion, by total gene synthetic suddenly change, the double-strand break reparation, like that.Including, but is not limited to relate to chimeric sudden change of constructing body is also included among the present invention.In one embodiment, sudden change can be by the sequence that includes, but is not limited to of naturally occurring molecule or the naturally occurring molecule through changing or suddenling change, and sequence compares, physical properties, secondary, three grades or quaternary structure, crystalline structure or the like Given information is instructed.
Being seen herein article and the described program of case description.Other information see in the following open case of quoting herein and reference: people such as Ling, and Approaches to DNA mutagenesis:an overview, Anal Biochem.254 (2): 157-178 (1997); People such as Dale, Oligonucleotide-directed random mutagenesis using thephosphorothioate method, Methods Mol.Biol.57:369-374 (1996); Smith, In vitromutagenesis, Ann.Rev.Genet.19:423-462 (1985); Botstein ﹠amp; Shortle, Strategies andapplications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directedmutagenesis, Biochem.J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids ﹠amp; Molecular Biology(D.M.J compiles Springer Verlag, Berlin) (1987) for Eckstein, F. and Lilley; Kunkel, Rapid and efficient site-specific mutagenesiswithout phenotypic selection, Proc.Natl.Acad.Sci.USA82:488-492 (1985); People such as Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol.154,367-382 (1987); People such as Bass, Mutant Trp repressors with new DNA-bindingspecificities, Science242:240-245 (1988); Zoller ﹠amp; Smith, Oligonucleotide-directedmutagenesis using M1 3-derived vectors:an efficient and general procedure for theproduction of point mutations in any DNA fragment Nucleic Acids Res.10:6487-6500 (1982); Zoller﹠amp; Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors, Methods in Enzynol.100:468-500 (1983); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis:a simple method using two oligonucleotide primersand a single-stranded DNA template, Methods in Enzymol.154:329-350 (1987); People such as Taylor, The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nickedDNA, Nucl.Acids Res.13:8749-8764 (1985); People such as Taylor, The rapid generation ofoligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res.13:8765-8785 (1985); Nakamaye ﹠amp; Eckstein, Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl.Acids Res.14:9679-9698 (1986); People such as Sayers, 5 '-3 ' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. Acids Res.16:791-802 (1988); People such as Sayers, Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restriction endonucleases in the presenceof ethidium bromide, (1988) Nucl.Acids Res.16:803-814; People such as Kramer, The gapped duplexDNA approach to oligonucleotide-directed mutation construction, Nucl.Acids Res.12:9441-9456 (1984); Kramer ﹠amp; Fritz Oligonucleotide-directed construction of mutations viagapped duplex DNA, Methods in Enzymol.154:350-367 (1987); People such as Kramer, Improvedenzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directedconstruction of mutations, Nucl.Acids Res.16:7207 (1988); People such as Fritz, Oligonucleotide-directed construction of mutations:a gapped duplex DNA procedure withoutenzymatic reactions in vitro, Nucl.Acids Res.16:6987-6999 (1988); People such as Kramer, Differentbase/base mismatches are corrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E.coli, Cell38:879-887 (1984); People such as Carter, Improvedoligonucleotide site-directed mutagenesis using M13 vectors, Nucl.Acids Res.13:4431-4443 (1985); Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors, Methods in Enzymol.154:382-403 (1987); Eghtedarzadeh ﹠amp; Henikoff, Use ofoligonucleotides to generate large deletions, Nucl.Acids Res.14:5115 (1986); People such as Wells, Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, Phil. Trans.R.Soc.Lond.A 317:415-423 (1986); People such as Nambiar, Total synthesis and cloning of agene coding for the ribonuclease S protein, Science223:1299-1301 (1984); Sakmar and Khorana, Total synthesis and expression of a gene for the alpha-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin), Nucl.Acids Res.14:6361-6372 (1988); People such as Wells, Cassette mutagenesis:an efficient method for generation of multiplemutations at defined sites, Gene34:315-323 (1985); People such as Grundstrom, Oligonucleotide-directed mutagenesis by microscale ' shot-gun ' gene synthesis, Nucl.Acids Res.13:3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strand break repairin plasmids of Escherichia coli:a method for site-specific mutagenesis, Proc.Natl.Acad.Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology4:450-455 (1993); People such as Sieber, Nature Biotechnology, 19:456-460 (2001); W.P.C.Stemmer, Nature370,389-91 (1994); And I.A.Lorimer, I.Pastan, Nucleic Acids Res.23,3067-8 (1995).Other detailed descriptions to many aforesaid methods are found in Methods in EnzymologyIn the 154th volume, it is also described using effective control of various mutation method trouble shoot problems.
(for example) be applicable to the oligonucleotide that storehouse sudden change that for example makes synthetic enzyme or the present invention who changes tRNA suddenly change, normally basis is by Beaucage and Caruthers, Tetrahedron Letts.22 (20): 1859-1862, (1981) the solid phase phosphoramidite three ester methods of Miao Shuing, (for example) use as people such as Needham-VanDevanter, Nucleic Acids Res., the automated synthesizer described in the 12:6159-6168 (1984) comes chemosynthesis.
The present invention also relates to eukaryotic host cell, non-eukaryotic host cell and be used for by quadrature tRNA/RS in vivo incorporating the organism of alpha-non-natural amino acid into.Host cell is to use polynucleotide of the present invention, or comprise polynucleotide of the present invention (including, but is not limited to can be the carrier of the present invention of (for example) cloning vector or expression vector) construct body genetically engineered (include, but is not limited to, change shape, transduction or transfection).For example, quadrature tRNA, quadrature tRNA synthetic enzyme and the proteinic coding region for the treatment of derivatize are operationally to be connected with the genetic expression controlling elements that works in desired host cell.Carrier can (for example) be plasmid, cement grain, phage, bacterium, virus, exposed polynucleotide or in conjunction with the polynucleotide form.Carrier is introduced in cell and/or the microorganism by the standard method that comprises following method: electroporation (people such as Fromm, Proc.Natl.Acad.Sci.USA82,5824 (1985)), by virus vector infect, by in the matrix of beads or particle or on the surface (Klein et al., Nature327,70-73 (1987)) have the high speed trajectory osmosis of the small-particle of nucleic acid, and/or like that.
Host cell through through engineering approaches can be cultivated in habitual nutritional medium, is the described activity upgrading such as screening step, activation promotor or selection transformant when described nutritional medium is suitable.Described cell is cultivated in transgenic organism.Other comprise Freshney (1994) to (including, but is not limited to) cellular segregation and the useful reference of cultivation (for example, for follow-up separate nucleic acid) Culture of Animal Cells, a Manual of Basic Technique, the third edition, Wiley-Liss, New York and the reference of wherein quoting; People such as Payne (1992) Plant Cell and Tissue Culture in Liquid SystemsJohn Wiley﹠amp; Sons, Inc.New York, NY; Gamborg and Phillips (volume) (1995) Plant Cell, Tissue and Organ CultureFundamental Methods SpringerLab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (volume) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, FL.
Some methods of knowing that target nucleic acid is introduced in the cell are available, and any method can be used among the present invention.Described method comprises: infect (hereinafter further discussing) etc. with recipient's cell and the fusion of bacterium protoplastis, the electroporation that contain DNA, projectile body bombardment with virus vector.Bacterial cell can contain the quantity that DNA of the present invention constructs the plasmid of body in order to amplification.Make bacterial growth can separate (for example referring to Sambrook) by known the whole bag of tricks in the affiliated field to the plasmid in logarithmic phase and the bacterium.In addition, it is commercially available (for example referring to, EasyPrep being used for from the test kit of bacteria purification plasmid TM, FlexiPrep TM, all from GE Healthcare; StrataClean from Stratagene TMWith QIAprep from Qiagen TM).Further handle then through separating and the plasmid of purifying produces in order to transfectional cell or incorporates in the related vector other plasmids with the infection biological body into.Typical carrier contains transcribes and translates terminator, transcribes and rotaring intertranslating start sequence and the expression promoter that is used to regulate concrete target nucleic acid.Carrier optionally comprises the expressed sequence box, and described expressed sequence box contains at least one independently terminator sequence, permission sequence the box sequence of duplicating and the selectable marker that is used for prokaryotic system and eukaryotic system in eukaryote or prokaryotic organism or both (including, but is not limited to shuttle vectors).Carrier is suitable for duplicating in prokaryotic organism, eukaryote or both and merging.Referring to, Gillam ﹠amp; Smith, Gene8:81 (1979); People such as Roberts, Nature.328:731 (1987); Schneider, people such as E., Protein Expr.Purif.6 (1): 10-14 (1995); Ausubel, Sambrook, Berger (all are the same).Bacterium that is used to clone and the catalogue of phage (for example) are provided by ATCC, are for example published by ATCC The ATCC Catalogue of Bacteria and Bacteriophage(1992) people's (volume) such as Gherna provides.The theoretical point view that is used for sequencing, clone and molecular biological otherwise other base programs and basis also sees people (1992) such as Watson Recombinant DNA Second EditionScientific American Books, NY.In addition, basically any nucleic acid (with any nucleic acid through mark in fact, no matter standard or whether non-standard) can be custom or standard, and it is any source from various commercial source, (can be such as Midland Certified Reagent Company at World Wide Web Mcrc.comOn the Midland that buys, TX), The Great American Gene Company (can be at World WideWeb, Genco.comOn the Ramona that buys, CA), ExpressGen Inc. (can be at World Wide Web, Expressgen.comOn the Chicago that buys, IL), Operon Technologies Inc (Alameda, CA) and many other sources order.
XII. the expression in non-eukaryote and eukaryote
For obtaining the high level expression of clone hGH polynucleotide, the those skilled in the art will encode the polynucleotide subclone of hGH polypeptide of the present invention usually in expression vector, and this expression vector contains the ribosome binding site that instructs the strong promoter of transcribing, transcription/translation terminator and (if being used for the nucleic acid of coded protein) to be used for rotaring intertranslating start.The bacterium promotor that is fit to is for known to the those skilled in the art and be described in philtrums such as people such as (for example) Sambrook and Ausubel.
The bacterial expression system that is used for expressing hGH polypeptide of the present invention can obtain (people such as Palva, Gene 22:229-235 (1983) (including, but is not limited to) intestinal bacteria, bacillus kind, pseudomonas fluorescens, Pseudomonas aeruginosa, evil breath utmost point hair bacillus and Salmonellas; People such as Mosbach, Nature 302:543-545 (1983)).The test kit that is used for described expression system is commercially available.The eukaryotic expression system that is used for mammalian cell, yeast and insect cell is for known to the those skilled in the art and also be commercially available.Quadrature tRNA and aminoacyl tRNA synthetase in order to the situation of expressing hGH polypeptide of the present invention under, the host cell that is used to express is based on it and uses the ability of quadrature component and select.Exemplary host cell comprises gram-positive microorganism (Gram-positive bacteria) (including, but is not limited to bacillus pumilus (B.brevis), subtilis (B.subtilis) or streptomyces (Streptomyces)) and gram negative bacterium (Gram-negative bacteria) (intestinal bacteria, pseudomonas fluorescens, Pseudomonas aeruginosa, evil breath utmost point hair bacillus) and yeast and other eukaryotic cells.By described herein, can use to comprise the right cell of O-tRNA O-RS.
Eukaryotic host cell of the present invention or non-eukaryotic host cell provide the synthetic proteinic ability that comprises the alpha-non-natural amino acid of mass efficient.On the one hand, composition optionally comprises (including, but is not limited to) at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram or the above protein that comprises alpha-non-natural amino acid of 1 gram, or the amount that reached of available in vivo method for producing protein (detailed description to recombinant protein production and purifying is provided herein).On the other hand, protein is optionally to include, but is not limited to whenever rise to the protein of few 10 micrograms, whenever rise to the protein of few 50 micrograms, whenever rise to the protein of few 75 micrograms, whenever rise to the protein of few 100 micrograms, whenever rise to the protein of few 200 micrograms, whenever rise to the protein of few 250 micrograms, whenever rise to the protein of few 500 micrograms, whenever rise to few 1 milligram protein or whenever rise to few proteinic concentration more than 10 milligrams or 10 milligrams and be present in and include, but is not limited to cellular lysate, damping fluid, in the composition of medicine damping fluid or other liquid suspensions (include, but is not limited to, be include, but is not limited to any amount of volume of about 1nl between more than about 100L or the 100L).In eukaryotic cell or non-eukaryotic cell, the protein that comprises at least a alpha-non-natural amino acid of producing a large amount of (include, but is not limited to, may be with the amount that the additive method that includes, but is not limited in vitro translate is produced than usually bigger amount) is feature of the present invention.
Eukaryotic host cell of the present invention or non-eukaryotic host cell provide biosynthesizing to comprise the proteinic ability of mass efficient alpha-non-natural amino acid.For example, the protein that comprises alpha-non-natural amino acid can include, but is not limited to the concentration of following concentration at cell extract, cellular lysate, substratum, damping fluid and/or middle production like that: at least 10 micrograms per litre, at least 50 micrograms per litre, at least 75 micrograms per litre, at least 100 micrograms per litre, at least 200 micrograms per litre, at least 250 micrograms per litre or at least 500 micrograms per litre, at least 1 mg/litre, at least 2 mg/litre, at least 3 mg/litre, at least 4 mg/litre, at least 5 mg/litre, at least 6 mg/litre, at least 7 mg/litre, at least 8 mg/litre, at least 9 mg/litre, at least 10 mg/litre, at least 20,30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900 mg/litre, the 1g/ liter, the 5g/ liter, 10g/ rises or 10g/ rises above protein.
Expression system, cultivation and separate
HGH can express in comprising the many suitable expression system of (for example) yeast, insect cell, mammalian cell and bacterium.The description of exemplary expression system is provided in hereinafter.
Yeast
As used herein, term " yeast " comprises any yeast in each primary yeast of gene that can express coding hGH.Described yeast includes, but is not limited to, and ascosporogenous yeast (Endomycetale (Endomycetales)), basidiomycetes have spore yeast (basidiosporogenous yeast) and belong to the yeast of imperfect fungi (blastomycete (Blastomycetes)) group.Ascosporogenous yeast is divided into 2 sections, i.e. Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter comprises 4 subfamilies, be Schizosaccharomycoideae (Schizosaccharomycoideae) (for example, Schizosaccharomyces (genus Schizosaccharomyces)), Nadsonioideae (Nadsonioideae), Lipomycoideae and Saccharomycoideae (for example pichia belongs to (genera Pichia), Ke Lufeishi yeast belong (Kluyveromyces) and Saccharomycodes (Saccharomyces)).Basidiomycetes has the spore yeast to comprise basidiomycetes Leucosporidium (genera Leucosporidium), Rhodosporidium (Rhodosporidium), lock and throw yeast belong (Sporidiobolus), Filobasidiella (Filobasidium) and incense ashes plan lock load Pseudomonas (Filobasidiella).The yeast that belongs to imperfect fungi (blastomycete) group is divided into 2 sections, be Sporobolomycetaceae (Sporobolomycetaceae) (for example, Sporobolomyces (genera Sporobolomyces) and cloth are reined in and played spore yeast belong (Bullera)) and Cryptococcaceae (Cryptococcaceae) (for example Candida (genus Candida)).
For especially paying close attention to of using of the present invention is species in the following species: pichia belongs to, the Ke Lufeishi yeast belong, Saccharomycodes, Schizosaccharomyces, Hansenula (Hansenula), torulopsis (Torulopsis) and Candida, include, but is not limited to pichia pastoris phaff (P.pastoris), P.guillerimondii, yeast saccharomyces cerevisiae (S.cerevisiae), Carlsberg yeast (S.carlsbergensis), saccharomyces diastaticus (S.diastaticus), Douglas yeast (S.douglasii), Ke Luweier yeast (S.kluyveri), Nuo Bensi yeast (S.norbensis), ellipsoideus yeast (S.oviformis), Kluyveromyces lactis (K.lactis), Sumylact L yeast (K.fragilis), Candida albicans (C.albicans), maltose candiyeast (C.maltosa) and saccharomyces hansenii (H.polymorpha).
Selection is used to express the suitable yeast of hGH in those skilled in the art's state of the art.When selecting the yeast host that is used to express, suitable host can comprise that displaying has described host of (for example) good secretion capacity, lower protein degrading activity and total soundness.Yeast can be buied from the various sources that include, but is not limited to following source usually: Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, Universityof California (Berkeley, CA) and American Type Culture Collection (" ATCC ") (Manassas, VA).
Term " yeast host " or " yeast host cell " comprise the yeast that can be used as or use the recipient who acts on recombinant vectors or other transfer DNAs.Term comprises the filial generation of the original yeast host cell of accepting recombinant vectors or other transfer DNAs.Should be appreciated that because sudden change unexpected or that have a mind to, the filial generation of single parental cell can morphology add to the genome of original parent or total DNA aspect identical.Enough the filial generation similar in appearance to the parental cell of the parent that remains to be characterized by the relevant nature that the nucleotide sequence such as coding hGH exists is included in the filial generation of described definition indication.
Developed the expression and the commentaries on classics shape carrier that comprise extrachromosomal replication or merge carrier, be used for changeing shape to many yeast hosts.For example, developed expression vector (people such as Sikorski, GENETICS (1989) 122:19 that is used for yeast saccharomyces cerevisiae; People such as Ito, J.BACTERIOL. (1983) 153:163; People such as Hinnen, PROC.NATL.ACAD.SCI.USA (1978) 75:1929); The expression vector (people such as Kurtz, MOL.CELL.BIOL. (1986) 6:142) that is used for Candida albicans; The expression vector (people such as Kunze, J.BASIC MICROBIOL. (1985) 25:141) that is used for the maltose candiyeast; The expression vector (people such as Gleeson, J.GEN.MICROBIOL. (1986) 132:3459 that are used for saccharomyces hansenii; People such as Roggenkamp, MOL.GENETICS AND GENOMICS (1986) 202:302); Be used for Sumylact L zymic expression vector (people such as Das, J.BACTERIOL. (1984) 158:1165); The expression vector (people such as De Louvencourt, J.BACTERIOL. (1983) 154:737 that are used for Kluyveromyces lactis; People such as Van den Berg, BIOTECHNOLOGY (NY) (1990) 8:135); The expression vector (people such as Kunze, J.BASIC MICROBIOL. (1985) 25:141) that is used for P.guillerimondii; The expression vector (United States Patent (USP) the 5th, 324,639 that are used for pichia pastoris phaff; 4,929,555; With 4,837, No. 148; People such as Cregg, MOL.CELL.BIOL. (1985) 5:3376); The expression vector (people such as Beach, NATURE (1982) 300:706) that is used for schizosaccharomyces pombe (Schizosaccharomyces pombe); Be used to separate fat Ye Luoweiya yeast (Y.lipolytica); The expression vector of Aspergillus nidulans (A.nidulans) (people such as Ballance, BIOCHEM.BIOPHYS.RES.COMMUN. (1983) 112:284-89; People such as Tilburn, GENE (1983) 26:205-221; With people such as Yelton, PROC.NATL.ACAD.SCI.USA (1984) 81:1470-74); The expression vector (Kelly and Hynes, EMBOJ. (1985) 4:475-479) that is used for melanomyces (A.niger); The expression vector (EP 0244234) that is used for Trichodermareesei (T.reesia); With the expression vector (WO 91/00357) that is used for such as the filamentous fungus of Neurospora (Neurospora), Penicillium (Penicillium), the curved mould genus of neck (Tolypocladium), each reference is to incorporate this paper by reference into.
The control sequence of yeast vector is for known to the those skilled in the art and include, but is not limited to, from the promoter region such as the gene of following gene: alcoholdehydrogenase (ADH) (EP 0 284 044); Enolase; Glucokinase; Glucose-6-phosphate isomerase; Glyceraldehyde-3-phosphate-desaturase (GAP or GAPDH); Hexokinase; Phosphofructokinase; The 3-phoshoglyceric acid mutase; And pyruvate kinase (PyK) (EP 0 329 203).The yeast PHO5 gene of coding acid phosphatase also can provide effective promoter sequence people such as (, PROC.NATL.ACAD.SCI.USA (1983) 80:1) Miyanohara.Other promoter sequences that are fit to that use for yeast host can comprise glycerol 3-phosphate acid kinase people such as (, J.BIOL.CHEM. (1980) 255:12073) Hitzeman; With other glycolytic enzymes, such as pyruvic carboxylase, triosephosphate isomerase and phosphoglucose isomerase (people such as Holland, Biochemistry (1978) 17:4900; People such as Hess, J.Adv.ENZYME REG. (1969) 7:149) promotor.Induced Yeast promoter with extra advantage of transcribing of controlling by growth conditions can comprise alcoholdehydrogenase 2; Different cell pigment C; Acid phosphatase; Metallothionein(MT); Glyceraldehyde-3-phosphate dehydrogenase; The degrading enzyme relevant with nitrogen metabolism; Promoter region with the enzyme of undertaking the application of maltose and semi-lactosi.The carrier and the promotor that are fit to that are applicable to yeast expression are described among the EP 0073657 in addition.
Yeast is strengthened son and also can be used with Yeast promoter.In addition, synthetic promoter also can play Yeast promoter.For example, the upstream activation sequences (UAS) of Yeast promoter can engage with the transcription activating zone of another Yeast promoter, produces synthetic hybrid promoters.The example of described hybrid promoters comprises that the ADH that is connected with GAP transcription activating zone regulates sequence.Referring to United States Patent (USP) the 4th, 880,734 and 4,876, No. 197.The included promotor of other examples of hybrid promoters is by forming with the adjusting sequence such as ADH2, GAL4, GAL10 or the PHO5 gene of the glycolytic enzyme gene transcription activating area combination of GAP or PyK.Referring to EP 0 164 556.In addition, Yeast promoter can comprise the naturally occurring promotor in the non-yeast source with ability that combining yeast RNA polymerase and initiation transcribe.
Other controlling elementss that can comprise the part Yeast expression carrier comprise (for example) terminator (people such as Holland, J.BIOL.CHEM. (1981) 256:1385) from GAPDH or enolase gene.In addition, the copy source from 2 μ plasmid-deriveds is applicable to yeast.Be applicable to that the selection gene that zymic is fit to is the trp1 gene that is present in the yeast plasmid.Referring to, people such as Tschumper, GENE (1980) 10:157; People such as Kingsman, GENE (1979) 7:141.The trp1 gene is provided as the selectable marker that does not have the yeast mutation bacterial strain that ability grows in tryptophane.Similarly, the yeast strain (ATCC 20,622 or 38,626) of scarce Leu2 is to replenish by the known plasmid that has the Leu2 gene.
It is known to the those skilled in the art that exogenesis DNA is introduced method in the yeast host, and generally includes (but being not limited to) with the spheroplast of alkaline kation processing or the commentaries on classics shape of complete yeast host cell.For example, zymic changes shape can be according to people such as Hsiao, people such as PROC.NATL.ACAD.SCI.USA (1979) 76:3829 and VanSolingen, and the method described in J.BACT. (1977) 130:946 is carried out.Yet, such as the additive method that is used for DNA is introduced cell that merges by nuclear injection, electroporation or protoplastis, also can be by people such as SAMBROOK, the general description among the MOLECULAR CLONING:A LAB.MANUAL (2001) is used.Then, can use and be the standard technique culturing yeast host cell known to the those skilled in the art.
Be used at the proteinic additive method of yeast host cell expressing heterologous for known to the those skilled in the art.Usually referring to, No. the 20020055169th, U.S. patent application case, United States Patent (USP) the 6th, 361,969; 6,312,923; 6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; With 5,089, No. 398; Patent RE37 reviews in the U.S., and 343 and RE35, No. 749; PCT publication application case WO 99/078621; WO 98/37208; With WO 98/26080; European patent application EP 0 946 736; EP 0 732 403; EP 0 480 480; WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; With EP 0 164 556, described patent is to incorporate this paper by reference into.Again referring to people such as Gellissen, ANTONIE VAN LEEUWENHOEK (1992) 62 (1-2): 79-93; People such as Romanos, YEAST (1992) 8 (6): 423-488; Goeddel, METHODS IN ENZYMOLOGY (1990) 185:3-7, each reference is to incorporate this paper by reference into.
The yeast host bacterial strain is in the amplification stage, uses to the standard feed batch fermentation method known to the those skilled in the art, grows in fermentor tank.Fermentation process can utilize the difference of the pattern of path or expression control to revise owing to the carbon of concrete yeast host.For example, the fermentation of Saccharomycodes yeast host can need single glucose charging, compound nitrogen source (for example casein hydrolysate) and vitamin fortification repeatedly.On the contrary, the methylotrophic yeast pichia pastoris phaff can need glycerine, methyl alcohol and trace minerals charging, but only needs simple ammonium (nitrogen) salt to reach optimum growh and expression.Referring to (for example), United States Patent (USP) the 5th, 324, No. 639; People such as Elliott, J.PROTEIN CHEM. (1990) 9:95; With people such as Fieschko, BIOTECH.BIOENG. (1987) 29:1113.
Yet described fermentation process can have some common trait that is independent of the used yeast host strain.For example, the growth limitation nutrition that is generally carbon can be added to during the amplification stage in the fermentor tank to allow maximum growth.In addition, fermentation process uses usually through designing the fermention medium with carbon, nitrogen, basic salt, phosphorus and other micro-nutrients (VITAMIN, trace minerals and salt etc.) that contains q.s.The case description that is suitable for the fermention medium that belong to use for pichia is in United States Patent (USP) the 5th, 324, and 639 and 5,231, in No. 178, described patent is to incorporate this paper by reference into.
Insect cell through baculovirus infection
Term " insect host " or " insect host cell " refer to the insect that can be used as or use the recipient who acts on recombinant vectors or other transfer DNAs.Term comprises the filial generation of transfected protentomon host cell.Should be appreciated that because sudden change unexpected or that have a mind to, the filial generation of single parental cell can morphology add to the genome of original parent or total DNA aspect identical.Enough the filial generation similar in appearance to the parental cell of the parent that remains to be characterized by the relevant nature that the nucleotide sequence such as coding hGH exists is included in the filial generation of described definition indication.
Be used to express being chosen as known to the those skilled in the art of suitable insect cell of hGH.Some insect species fully are described in the affiliated field and are commercially available, comprise Aedes aegypti (Aedes aegypti), silkworm (Bombyxmori), fruit bat (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) and cabbage looper (Trichoplusia ni).When selecting the insect host that is used to express, suitable host can comprise that displaying has described host of (especially) good secretion capacity, lower protein degrading activity and total soundness.Insect can be buied from the various sources that include, but is not limited to following source usually: Insect Genetic Stock Center, Department of Biophysicsand Medical Physics, University of California (Berkeley; CA); With American Type CultureCollection (" ATCC ") (Manassas, VA).
Usually, comprise transfer vector, be generally bacterial plasmid, the suitable restriction site that it contains the genomic fragment of baculovirus and is used to insert heterologous gene to be expressed through the component of the insect expression system of baculovirus infection; Have with transfer vector in the wild-type baculovirus (its allow heterologous gene the homologous recombination in the baculovirus genome) of baculovirus specific fragment homologous sequence; With suitable insect host cell and growth medium.Be used for construction carrier, transfectional cell, select the bacteriolyze spot, cell is grown at culture, suchlike material, method and technology are available for handbook known and that describe described technology in affiliated field.
After inserting heterologous gene in the transfer vector, in the transfected insect host cell of recombinating therein to carrier and viral genome of carrier and wild-type virus genome.Expression is through recombinant virus and the identification and the purification of Recombinant bacteriolyze spot of packing.The material and the method that are used for baculovirus/insect cell expression system are so that (Carlsbad, kit form CA) is commercially available from (for example) Invitrogen Corp..Described technology is generally known to the those skilled in the art and all is described in the SUMMERS AND SMITH that incorporates this paper by reference into, among the TEXAS AGRICULTURALEXPERIMENT STATION BULLETIN NO.1555 (1987).Again referring to, RICHARDSON, 39METHODS IN MOLECULAR BIOLOGY:BACULOVIRUS EXPRESSION PROTOCOLS (1995); People such as AUSUBEL, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM:A LABORATORY GUIDE (1992); With, people such as O ' REILLY, BACULOVIRUS EXPRESSION VECTORS:A LABORATORYMANUAL (1992).
In fact, use the production of various heterologous proteins of baculovirus/insect cell expression system as known to the those skilled in the art.Referring to (for example), United States Patent (USP) the 6th, 368,825; 6,342,216; 6,338,846; 6,261,805; 6,245,528,6,225,060; 6,183,987; 6,168,932; 6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676; 5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220; 5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290, No. 686; WO 02/06305; WO 01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO 99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO 97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO 93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO 90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO 89/01037; WO 88/07082, and described patent is incorporated this paper by reference into.
The carrier that is applicable to baculovirus/insect cell expression system is known and comprises insect expression and the transfer vector that (for example) obtained by baculovirus Autographa californica multicapsid nucleopolyhedrosisvirus (Autographacalifornica) nuclear polyhedrosis virus (AcNPV) that it is helper virus (helper) independence, virus expression carrier in affiliated field.The virus expression carrier that is obtained by described system uses the strong virus polyhedrin gene promoter to drive expression of heterologous genes usually.Usually referring to, people such as O ' Reilly, BACULOVIRUS EXPRESSION VECTORS:A LABORATORYMANUAL (1992).
Before alien gene being inserted in the shaft-like viral genome, the said components that will comprise promotor, introduction (if desired), the encoding sequence of being paid close attention to and transcription termination sequence usually is assembled into middle dislocation and constructs body (transfer vector).Often the centre dislocation is constructed body maintain such as can stable maintenance in replicon such as the extrachromosome element among the host of bacterium (for example plasmid).Therefore replicon will have dubbing system, allows that it is maintained to be used for the host who is fit to that clones and increase.More particularly, plasmid can contain (amp) gene and be used for selecting and the copy source of breeding intestinal bacteria of polyhedrin polyadenylation signal (Miller, Ann.Rev.Microbiol. (1988) 42:177) and the anti-penbritin of protokaryon (ampicillin).
The transfer vector a kind of commonly used that is used for alien gene is introduced AcNPV is pAc373.Also designed to many other carriers known to the those skilled in the art, comprised (for example) pVL985, it changes over ATT with the polyhedrin initiator codon from ATG, and introduces BamHI clone positions at 32 base pair places in ATT downstream.Referring to, Luckow and Summers, VIROLOGY 170:31 (1989).Other commercially available carriers comprise (for example) PBlueBac4.5/V5-His; PBlueBacHis2; PMelBac; PBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).
After inserting heterologous gene, with transfer vector and wild-type baculovirus genome cotransfection in the insect cell host.Be used for allogeneic dna sequence DNA introduce method in the desired position of shaft-like virus-virus in affiliated field for known.Referring to, SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATIONBULLETIN NO.1555 (1987); People such as Smith, MOL.CELL.BIOL. (1983) 3:2156; Luckow andSummers, VIROLOGY (1989) 170:31.For example, insert to can be and recombinate, be inserted in the gene such as polyhedron gene by the homology crossover; Insert also to can be and be inserted in the restriction enzyme position that is engineered desired baculovirus gene.Referring to, people such as Miller, BiOESSAYS (1989) 11 (4): 91.
Transfection can be finished by electroporation.Referring to, TROTTER AND WOOD, 39METHODS INMOLECULAR BIOLOGY (1995); Mann and King, J.GEN.VIROL. (1989) 70:3501.Perhaps, liposome can be used recombinant expression vector and baculovirus transfection insect cell.Referring to (for example), people such as Liebman, BIOTECHNIQUES (1999) 26 (1): 36; People such as Graves, BIOCHEMISTRY (1998) 37:6050; People such as Nomura, J.BIOL.CHEM. (1998) 273 (22): 13570; People such as Schmidt, PROTEINEXPRESSION AND PURIFICATION (1998) 12:323; People such as Siffert, NATURE GENETICS (1998) 18:45; People such as TILKINS, CELL BIOLOGY:A LABORATORY HANDBOOK 145-154 (1998); People such as Cai, PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263; People such as Dolphin, NATURE GENETICS (1997) 17:491; People such as Kost, GENE (1997) 190:139; People such as Jakobsson, J.BIOL.CHEM. (1996) 271:22203; People such as Rowles, J.BIOL.CHEM. (1996) 271 (37): 22376; People such as Reverey, J.BIOL.CHEM. (1996) 271 (39): 23607-10; People such as Stanley, J.BIOL.CHEM. (1995) 270:4121; People such as Sisk, J.VlROL. (1994) 68 (2): 766; With people such as Peng, BIOTECHNIQUES (1993) 14 (2): 274.Commercially available liposome comprises (for example),
Figure A20058004446300831
With
Figure A20058004446300832
(Invitrogen, Corp., Carlsbad, CA).In addition, can use calcium phosphate transfection.Referring to, TROTTER AND WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990) 18 (19): 5667; With Mann and King, J.GEN.VIROL. (1989) 70:3501.
Rhabdovirus expression vector contains bacilliform virus promoter usually.Bacilliform virus promoter is anyly can and cause the dna sequence dna that encoding sequence (for example structure gene) downstream (3 ') is transcribed into mRNA in conjunction with shaft-like viral rna polymerase.Promotor will have and be usually located at the transcription initiation zone of approaching 5 of encoding sequence ' end most.Described transcription initiation zone generally includes RNA polymerase combining site and transcription initiation position.Bacilliform virus promoter also can have to be called strengthens the second sub territory, if exist, it is usually away from structure gene so.In addition, express and can be modulability or for constitutive character.
The structure gene of fully transcribing in the infection round-robin later stage provides especially effectively promoter sequence.Example comprises by coding viral polyhedron proteinic gene (people such as FRIESEN, The Regulation of Baculovirus GeneExpression in THE MOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and O 155 476) and the sequence that obtains of coding p10 proteinic gene people such as (, J.GEN.VIROL. (1988) 69:765) Vlak.
The rhabdovirus expression vector that newly forms is packaged into infects in the recombinant baculovirus and subsequently, the bacteriolyze spot that can grow by the technology purifying known to the those skilled in the art.Referring to, people such as Miller, BIOESSAYS (1989) 11 (4): 91; SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENTSTATION BULLETIN No.1555 (1987).
Developed the recombination rhabdovirus expression vector that is used for infecting some insect cells.For example, developed the recombinant baculovirus that is particularly useful for Aedes aegypti (ATCC CCL-125 number), silkworm (ATCC CRL-8910 number), fruit bat (No. the 1963rd, ATCC), fall army worm and cabbage looper.Referring to, Wright, NATURE (1986) 321:718; People such as Carbonell, J.VIROL. (1985) 56:153; People such as Smith, MOL.CELL.BIOL. (1983) 3:2156.Usually referring to, people such as Fraser, I NV ITROCELL.DEV.BIOL. (1989) 25:225.More particularly, the clone that is used for the rhabdovirus expression vector system generally includes (but being not limited to) Sf9 (fall army worm) (ATCC CRL-1711 number), Sf21 (fall army worm) (Invitrogen Corp., Cat. 11497-013 number (Carlsbad, CA)), Tri-368 (cabbage looper) and High-Five TMBTI-TN-5B1-4 (cabbage looper).
The cell and the substratum that are used at direct expression of baculovirus/expression and amalgamation and expression heterologous polypeptide are commercially available, and cell culture technology is to be generally known to the those skilled in the art.
Intestinal bacteria, pseudomonas kind and other prokaryotic organism
The bacterial expression technology is known to the those skilled in the art.Variety carrier can obtain being used for host bacterium.Carrier can be the low or high multi-copy vector of single copy.Carrier can be used for the clone and/or express.In view of about the commercial availability of carrier, many carriers and even the abundant document of the handbook of carrier and its restriction figure and feature is described, do not need extensive discussions at this.As is well known, carrier be usually directed to allow the marker selected, described marker can provide the cytotoxic agent resistivity, former nutrition or immunity.Usually, there are a plurality of markers that different characteristics is provided.
The bacterium promotor is any dna sequence dna that can encoding sequence (for example structure gene) downstream (3 ') be transcribed into mRNA in conjunction with bacteria RNA polysaccharase and initiation.Promotor will have and be usually located at the transcription initiation zone of approaching 5 of encoding sequence ' end most.Described transcription initiation zone generally includes RNA polymerase combining site and transcription initiation position.The promotor of bacterium also can have second territory that is called operon, and it can the synthetic contiguous RNA polymerase combining site that begins to locate of overlapping RNA.Operon allows that negativity regulates (derivable) and transcribe, because gene repression albumen can and and then suppress transcribing of specific gene in conjunction with operon.Constitutive character is expressed and can be taken place when the negativity regulatory element that does not exist such as operon.In addition, positivity is regulated and can be reached by the sub-protein binding sequence of gene activation, if exist, it approaches RNA polymerase binding sequence (5 ') usually most so.The proteinic example of gene activation is metabolite activated protein (CAP), and it helps to cause transcribing in lac operon Zai Da Intestinal Escherichia (E.coli) people such as [, ANNU.REV.GENET. (1984) 18:173] Raibaud.Therefore expression through regulating can be positively charged or negative, and then strengthens or reduce and transcribe.
The sequence of coding metabolic pathway enzyme provides especially effectively promoter sequence.Example comprises the promoter sequence that is obtained by the carbohydrate metabolism enzyme such as semi-lactosi, lactose (lac) [people such as Chang, NATURE (1977) 198:1056] and maltose.Other examples comprise promoter sequence [people such as Goeddel, Nuc.ACIDS RES. (1980) 8:4057 that is obtained by the biosynthetic enzyme such as tryptophane (trp); People such as Yelverton, NUCL.ACIDS RES. (1981) 9:731; United States Patent (USP) the 4th, 738, No. 921; The open case the 036776th of European patent and No. 121775, it is to incorporate this paper by reference into].People such as g-lactamase (bla) promoter systems [Weissmann (1981) " The cloning of interferon and othermistakes. " In Interferon 3 (I.Gresser volume)], phage PL[Shimatake, NATURE (1981) 292:128] and T5[United States Patent (USP) the 4th, 689, No. 406] promoter systems also provides effective promoter sequence.The preferred method of the present invention utilization is induced high-caliber hGH such as the strong promoter of T7 promotor.The example of described carrier is for known to the those skilled in the art and comprise from the pET29 series of Novagen and be described in pPOP carrier among the WO99/05297.Described expression system produces high-caliber hGH in the host, and can not endanger host cell survivability or growth parameter(s).PET19 (Novagen) is a known another kind of carrier in affiliated field.
In addition, the synthetic promoter of non-natural existence also plays the bacterium promotor.For example, the transcription-activating sequence of bacterium promotor or phage promoter can engage with the operon sequence of another bacterium promotor or phage promoter, produces synthetic hybrid promoters [United States Patent (USP) the 4th, 551, No. 433, described patent is to incorporate this paper by reference into].For example, the tac promotor is the hybridization trp-lac promotor of being made up of trp promotor and lac operon sequence of regulating by the lac repressor [people such as Amann, GENE (1983) 25:167; People such as de Boer, PROC.NATL.ACAD.SCI. (1983) 80:21].In addition, the bacterium promotor can comprise the naturally occurring promotor in the non-bacterial source with the ability of transcribing in conjunction with bacteria RNA polysaccharase and initiation.The naturally occurring promotor in non-bacterial source also can with compatible RNA polymerase coupling to produce the high level expression of some genes in prokaryotic organism.Bacterium Phosphoric acid esterase (bacteriophase) t7 rna polymerase/promoter systems is example [people such as Studier, J.MOL.BIOL. (1986) 189:113 through the promoter systems of coupling; People such as Tabor, Proc Natl.Acad.Sci. (1985) 82:1074].In addition, hybrid promoters also can be made of (No. the 267851st, the open case of European patent) phage promoter and intestinal bacteria operon zone.
Except that the function on subsequence, effectively ribosome binding site also is applicable to the expression of alien gene in prokaryotic organism.In intestinal bacteria, ribosome binding site is called Shine-Dalgarno (SD) sequence and the length that comprises initiator codon (ATG) and be positioned 3-11 Nucleotide upstream of initiator codon is the sequence [people such as Shine, NATURE (1975) 254:34] of 3-9 Nucleotide.Think of the pairing of SD sequence by the base between 3 ' end of SD sequence and intestinal bacteria 16S rRNA, and promote mRNA and ribosomal combination the [people " Genetic signalsand nucleotide sequences in messenger RNA " such as Steitz, In Biological Regulation and Development:Gene Expression (Ed.R.F.Goldberger, 1979)].Express eukaryotic gene and prokaryotic gene people " Expression of cloned genes in Escherichia coli " such as [, Molecular Cloning:A Laboratory Manual, 1989] Sambrook with weak ribosome binding site.
Term " host bacterium " or " bacterial host cell " refer to the bacterium that can be used as or use the recipient who acts on recombinant vectors or other transfer DNAs.Term comprises the filial generation of the primitive bacteria host cell of transfection.Should be appreciated that because sudden change unexpected or that have a mind to, the filial generation of single parental cell can morphology be complemented at the genome of original parent or total DNA aspect identical.Enough the filial generation similar in appearance to the parental cell of the parent that remains to be characterized by the relevant nature that the nucleotide sequence such as coding hGH exists is included in the filial generation of described definition indication.
Be used to express being chosen as known to the those skilled in the art of suitable host bacteria of hGH.When selecting the host bacterium that is used to express, suitable host can comprise that displaying has described host that (especially) good inclusion body forms ability, lower protein degrading activity and total soundness.Host bacterium can be buied from the various sources that include, but is not limited to following source usually: Bacterial Genetic Stock Center, Department of Biophysics and MedicalPhysics, University of California (Berkeley, CA); With American Type Culture Collection (" ATCC ") (Manassas, VA).Bacterium that is obtained by K bacterial strain (for example W3110) or the bacterium that is obtained by B bacterial strain (for example BL21) are used in industrial fermentation/medicine fermentation usually.Described bacterial strain is especially effective because its growth parameter(s) be very know with firm.In addition, described bacterial strain right and wrong are pathogenic, its commercial be important to security and environment reason.In one embodiment, escherichia coli host is the bacterial strain that includes, but is not limited to the DH10B of DH10B (fis).Other examples of the escherichia coli host that is fit to include, but is not limited to the bacterial strain of BL21, DH10B or derivatives thereof.In another embodiment, escherichia coli host is the bacterial strain of W3110.The recombinant host cell bacterial strain can be modified to optimize desired feature by genetic mutation.For example, the host cell bacterial strain can be genetically modified to regulate the expression such as the metabolic important gene of the described gene that relates to carbon source metabolism, amino acid metabolism or proteolytic enzyme production.Described gene can be through sudden change to reduce, to increase, to reject or to knock in expression in desired host strain.For example, bacterial strain W3110 can be modified to be implemented in the genetic mutation in the metabolic gene that one or more that include, but is not limited to the araB gene relate to pectinose (arabinose).For example, bacterial strain can be modified to realize genetic mutation or to reject other genes.Sudden change or the method for rejecting gene are known to the those skilled in the art.Bacterial strain also can be through sudden change to regulate the intrinsic protein enzymic activity with the production that increases total length hGH and/or minimize external chemical inhibitor being added to the needs of proteolytic enzyme.Other host cell bacterial strains include, but is not limited to BL21.In another embodiment of method of the present invention, escherichia coli host is the negative bacterial strain of proteolytic enzyme, includes, but is not limited to OMP-and LON-.The host cell bacterial strain can be the pseudomonas kind, includes, but is not limited to pseudomonas fluorescens, Pseudomonas aeruginosa and evil breath utmost point hair bacillus.The pseudomonas fluorescens biovariety 1 of known called after bacterial strain MB 101 is applicable to recombinant production and can be used for the therapeutic protein production technique.The example of pseudomonas expression system comprises can host strain (can be at World Wide Web, Dow.comOn the Midland that buys, MI) available from the system of Dow Chemical Company.The United States Patent (USP) the 4th, 755,465 and 4,859 of incorporating this paper by reference into No. 600, is described the purposes of pseudomonas strain as the host cell that is used for hGH production.
In case the recombinant host cell bacterial strain is set up (that is to say, express to construct body be introduced in the host cell and have correct expression to construct the host cell of body separated), just cultivate recombinant host cell being suitable for producing under the condition of hGH.Conspicuous as the those skilled in the art, cultivate the method for recombinant host cell bacterial strain and will construct the character of body and the identity of host cell is decided on the expression that is utilized.The method that is usually used as known to the those skilled in the art is cultivated the recombinant host bacterial strain.Recombinant host cell normally contains VITAMIN, amino acid, somatomedin and other at the assimilable source that contains carbon, nitrogen and inorganic salt and (optionally) to be cultivated for the protein known to the those skilled in the art in the liquid nutrient medium of fill-in and cultivates.Being used for the substratum of optimum growh or feed composition and/or nutrient need can be owing to different recombinant host cells and/or because littler more massive relatively preparation and difference.For example, needed trace-metal or VITAMIN can and/or use substituting host cell to change with the growth conditions change.For optimizing the production of hGH polypeptide, being suitable for the inductive condition can look employed recombinant host cell, express and construct body and/or to changing such as the modification that suddenlys change that host cell carries out, include, but is not limited to be used for the change of inductive pectinose level.Pectinose level in the fermentation can be between about 0.0001% to about 0.1%, include, but is not limited to 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.0095%, 0.009%, 0.0085%, 0.008%, 0.0075%, 0.007%, 0.0065%, 0.006%, 0.0055%, 0.005%, 0.0045%, 0.004%, 0.0035%, 0.003%, 0.0025%, 0.002%, 0.0015%, 0.001%, 0.00095%, 0.0009%, 0.00085%, 0.0008%, 0.00075%, 0.0007%, 0.00065%, 0.0006%, 0.00055%, 0.0005%, 0.00045%, 0.0004%, 0.00035%, 0.0003%, 0.00025%, 0.0002%, 0.00015%, 0.0001%.In certain embodiments, the pectinose level is between 0.0005% and 0.05%.In certain embodiments, the pectinose level is between 0.001% to 0.02%.Also can carry out change so that higher cell harvesting density to be provided; Can carry out the step that includes, but is not limited to add second charging.The liquid nutrient medium that is used to cultivate host cell can optionally contain microbiotic or the anti-mycotic agent that prevents the undesirable microorganism growth and/or include, but is not limited to antibiotic compound to select to contain the host cell of expression vector.Recombinant host cell can be cultivated in batches or with continuous form, and the cell harvesting (under the situation of variant hGH cell inner accumulated) or the collection of culture supernatants simultaneously is form in batches or continuously.For producing in prokaryotic host cell, batch culture and cell harvesting are preferred.
Can carry out and regulate suppress, suppress continuously or induce inhibition.To it will be apparent to those skilled in the art that, non-naturally encoded amino acid can be added in the cell culture at a plurality of different times of cell growing period, or can be at cell growing period continued presence.Add one or more non-naturally encoded amino acid that are used for incorporating into hGH and can induce before hGH expresses generation when inducing or inducing after by host cell.In one embodiment, non-naturally encoded amino acid is to add before inducing the hGH expression.In one embodiment, non-naturally encoded amino acid is roughly to add 1 hour the time before inducing.In another embodiment, non-naturally encoded amino acid is to exist at the cell growing period.
Express the recombinant host cell of hGH, no matter whether be solubility, through excretory or insoluble, can multiple volume of culture grow.Method of the present invention is obeyed little laboratory scale volume of culture and large-scale industry scale volume.To it will be apparent to those skilled in the art that, the method for the present invention of Jie Shiing can be elevated to big volume of culture herein.The large-scale industry volume of culture can have broad range, for example each since more than 1 liter or 1 liter to hundreds of liters, thousands of liter, 5000 liters, 10,000 liters, 20,000,30,000 liter, 40,000 liters, 50,000 liters, up to more than 100,000 liters or 100,000 liters.When producing extensive volume, can be necessary and be conspicuous the those skilled in the art to the modification of some steps of method.
HGH of the present invention is purified after normally in being expressed in recombination system.HGH can be by known the whole bag of tricks in the affiliated field from host cell or substratum purifying.The hGH that results from the bacterial host cell can be (being the inclusion body form) indissoluble or insoluble.Under the situation of insoluble protein, protein can be collected and can then carry out the homogenization of cell in addition from the host cell lysate by centrifugal.Under the proteinic situation of indissoluble, can add include, but is not limited to polymine (PEI) compound to induce partly soluble proteinic precipitation.Then, the sedimentary protein of institute can be collected easily by centrifugal.Can use to be the whole bag of tricks known to the those skilled in the art, recombinant host cell is cracked or homogenize to discharge inclusion body in cell.Can use and include, but is not limited to that the enzymatic cell is cracked, supersound process, Du Ensi (dounce) homogenization or high pressure discharge the cracked technology of knowing, and carries out the cracked or homogenization of host cell.In an embodiment of method of the present invention, the high pressure release tech is used so that e. coli host cell is cracked to discharge the inclusion body of hGH.
Then, known multiple suitable solubilizing agent any in the field makes insoluble or heavy hGH dissolving of forming sediment under can using.HGH can dissolve with urea or Guanidinium hydrochloride.The volume of the dissolved hGH of institute is minimized so that can manageable batch weight easy to use produce in enormous quantities.Described factor can be important in large-scale industry is set, and wherein recombinant host can volume be the batch growth that is thousands of liters.In addition,, when being particularly useful for human medicinal use, if possible, should avoid so damaging machine and container when setting with large-scale industry when making hGH, or the not good chemical of protein itself.Show that in the method for the invention the denaturing agent urea of milder can make the dissolving of hGH inclusion body in order to replace harsher denaturing agent Guanidinium hydrochloride.The use meeting of urea significantly reduces the risk to the damage of the stainless steel equipment that is utilized in the manufacturing of hGH and purifying process, make the dissolving of hGH inclusion body simultaneously effectively.
Under the proteinic situation of solubility hGH, can be secreted into hGH in the periplasmic space or be secreted in the substratum.In addition, solubility hGH may reside in the tenuigenin of host cell.Before carrying out purification step, may need to concentrate solubility hGH.For the standard technique known to the those skilled in the art can be in order to concentrate solubility hGH from (for example) cellular lysate or substratum.In addition, can be with so that host cell be cracked and discharge solubility hGH from the tenuigenin or the periplasmic space of host cell for the standard technique known to the those skilled in the art.
When producing hGH, can remove fusion sequence as fused protein.Removing of fusion sequence can be finished by enzymatic lysis or chemical cracking.The enzymatic of fusion sequence is removed the method that can use to known to the those skilled in the art and is finished.The selection that is used to remove the enzyme of fusion sequence will determine by the identity that merges, and as will be conspicuous to the those skilled in the art, reaction conditions will be stipulated by the selection of enzyme.Chemical cracking can use to known to the those skilled in the art, and the reagent that includes, but is not limited to cyanogen bromide, TEV proteolytic enzyme and other reagent is finished.Through cracked hGH can by for the method known to the those skilled in the art from through cracked fusion sequence purifying.As will be conspicuous to the those skilled in the art, described method will be determined by identity and the character of fusion sequence and hGH.The method that is used for purifying can include, but is not limited to spatial exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography or dialysis or its any combination.
Also can purifying hGH to remove DNA from protein soln.DNA can also can remove by using such as the nucleic acid precipitation agent generation precipitation of (but being not limited to) protamine sulfate by removing such as known any suitable method in the affiliated field of precipitation or ion exchange chromatography.Can use the well-known process that includes, but is not limited to centrifugal or filtering standard, separate hGH from the sedimentary DNA of institute.Removing of host's nucleic acid molecule is that hGH treats in order to the important factor in the setting for the treatment of the mankind therein, and method of the present invention is reduced to pharmaceutically acceptable level with host cell DNA.
Include, but is not limited to fermentor tank, shake the small-scale fermentation of bottle, fluidized bed bio-reactor, hollow-fiber bioreactor, rolling bottle culture systems and steel basin bioreactor system or the method for large scale fermentation also can be used for protein expression.Each method of described method can batch processes, charging-batch processes or continuous mode method are carried out.
Method standard under human hGH polypeptide of the present invention can use usually in the field reclaims.For example, substratum or cellular lysate can be through centrifugal or filter to remove cell debris.Can or be diluted to desired volume or filter thoroughly in the damping fluid that is fit to supernatant concentration and be used to be further purified to regulate preparation.The purifying of hGH polypeptide can comprise the deamidated form and the form through pruning of separating the hGH polypeptide variants from complete form.
Any program in the following exemplary sequence can be used for purifying hGH polypeptide of the present invention: affinity chromatography; Negatively charged ion or cation-exchange chromatography (use includes, but is not limited to DEAE SEPHAROSE); The silica chromatography; High performance liquid chromatography (HPLC); Anti-phase HPLC; Gel-filtration (use includes, but is not limited to SEPHADEX G-75); The hydrophobic interaction chromatography; Spatial exclusion chromatography; Metal chelate chromatography; Ultrafiltration/diafiltration; Ethanol sedimentation; Ammonium sulfate precipitation; Chromatofocusing; Displacement chromatography; Electrophoretic procedures (including, but is not limited to the isoelectrofocusing of preparation property), difference solubility (including, but is not limited to ammonium sulfate precipitation), SDS-PAGE or extraction method.
Protein of the present invention, include, but is not limited to comprise alpha-non-natural amino acid protein, comprise alpha-non-natural amino acid proteinic antibody, comprise combination of proteins collocation thing of alpha-non-natural amino acid or the like, can be according to the standard program of using with the those skilled in the art known to the those skilled in the art, partly or substantially purifying reaches uniformity.Therefore, polypeptide of the present invention can be by including, but is not limited to, ammonium sulfate or ethanol sedimentation, acid or alkali extraction method, column chromatography, affinity column chromatography method, negatively charged ion or cation-exchange chromatography, phosphocellulose chromatography method, hydrophobic interaction chromatography, hydroxyapatite chromatography method, Sugar receptors chromatography, gel electrophoresis are suchlikely reclaimed and purifying for any method in the whole bag of tricks known to the those skilled in the art.Can on demand when making correctly folding mature protein, use the protein refolding step.High performance liquid chromatography (HPLC), affinity chromatography or other methods that is fit to can be used for wherein requiring highly purified final purification step.In one embodiment, antibody through making with the opposing alpha-non-natural amino acid protein of alpha-non-natural amino acid (or comprise) can be used as purified reagent, include, but is not limited to the purified reagent of the proteinic purifying based on avidity that comprises one or more alpha-non-natural amino acids.In case on demand by purifying partly or reach uniformity, polypeptide just optionally is used for multiple function, include, but is not limited to as calibrating component, therapeutical agent, preventive, diagnostic reagent, research reagent and/or as the immunogen of antibody producing.
Except that other reference that this paper indicates, various purifying/protein folding method is known to the those skilled in the art, include, but is not limited to below with reference to the method for describing in the document: R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzyrnology Vol.182:Guide to Protein Purification,Academic Press, Inc.N.Y. (1990); Sandana, (1997) Bioseparation of Proteins, Academic Press, Inc.; People such as Bollag (1996) Protein Methods, second edition Wiley-Liss, NY; Walker, (1996) The Protein Protocols HandbookHumana Press, NJ, Harris and Angal, (1990) Protein Purification Applications:A Practical ApproachIRL Press at Oxford, Oxford, England; Harris and Angal, Protein Purification Methods:A Practical ApproachIRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification:Principles and Practice the 3rd VersionSpringer Verlag, NY; Janson and Ryden, (1998) Protein Purification:Principles, High Resolution Methods and Applications, second editionWiley-VCH, NY; And Walker (1998), Protein Protocols on CD-ROMHumana Press, NJ; The reference of wherein quoting.
In eukaryotic host cell or non-eukaryotic host cell, a protein with alpha-non-natural amino acid that generation is paid close attention to or an advantage of polypeptide are that protein or polypeptide are folded into its native conformation usually.Yet, in certain embodiments of the present invention, those skilled in the art will realize that protein can have the conformation of the desired conformation that is different from related polypeptide after synthetic, expression and/or purifying.In one aspect of the invention, expressed protein is optionally by sex change and subsequently by renaturation.Described processing is that known method is finished in the affiliated field of utilization, include, but is not limited to by chaperonin being added to protein or the polypeptide of being paid close attention to, by protein is dissolved in the chaotropic agent such as guanidine HCl, utilize protein disulfide-isomerase to wait and finish.
Generally speaking, need to make expressed polypeptide sex change and reduction and make polypeptide be folded into preferred conformation more subsequently occasionally.For example, can add guanidine, urea, DTT, DTE and/or chaperonin to paid close attention to translational product.The method that makes protein reduction, sex change and renaturation for known to the those skilled in the art (referring to, people (1993) such as above-mentioned reference and Debinski J.Biol.Chem.,268:14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4:581-585; With people such as Buchner, (1992) Anal.Biochem.,205:263-270).For example, people such as Debinski describes sex change and the reduction of inclusion body protein in guanidine-DTE.Protein can contain include, but is not limited in Sleep-promoting factor B and the arginic oxidation-reduction quality damping fluid of L-more folding.Folding reagent is flowed or otherwise move contacting with one or more polypeptide or other expression products, or vice versa.
Produce at protokaryon under the situation of hGH, therefore the hGH that produces can be false folding and therefore lack biological activity or have the biological activity of reduction.Proteinic biological activity can be recovered by " folding again ".Generally speaking, the hGH of false folding is by using (for example) one or more chaotropic agents (for example urea and/or guanidine) and can reduce the reductive agent (for example dithiothreitol (DTT), DTT or 2 mercapto ethanol, 2-ME) of disulfide linkage, makes polypeptide chain dissolving (wherein hGH also is insoluble), separates folding and reduction and folding again.Then, under the chaotropic agent of intermediate concentration, add oxygenant (for example oxygen, Gelucystine or cystamine), it can form disulfide linkage again.Under hGH can use in the field known standard method folding again, such as United States Patent (USP) the 4th, 511,502,4,511,503 and 4,512, the described method in No. 922.
After folding again, can be further purified hGH.The purifying of hGH can use and be the various technology known to the those skilled in the art, comprise hydrophobic interaction chromatography, spatial exclusion chromatography, ion-exchange chromatography, anti-phase high performance liquid chromatography, affinity chromatography, like that or its any combination and finishing.Extra purifying also can comprise drying or precipitate purified proteinic step.
After the purifying, hGH can be switched in the different damping fluids and/or be concentrated by any method in the known the whole bag of tricks in the affiliated field that includes, but is not limited to ultrafiltration, diafiltration and dialysis.Be provided as through the proteinic hGH of single purifying and can stand to assemble and precipitation.The multiple material of buffer exchange or concentrated polypeptide is known to the those skilled in the art.
Purified hGH can be at least 90% pure (pressing by the anti-phase high performance liquid chromatography, be RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, be that SDS-PAGE is measured) or it is at least 95% pure, at least 98% is pure, or at least 99% or 99% is above pure.No matter the definite numerical value of the purity of hGH, concerning as pharmaceutical prod or be used for such as with such as other processing of the keying action of the water-soluble polymers of PEG, hGH is enough pure.
Some hGH molecule can not exist other activeconstituentss or protein to be used as therapeutical agent when (being different from vehicle, supporting agent and stablizer, serum albumin, like that), or it can be compound with another kind of protein or polymkeric substance.
XIII. Purification process
Any step in the various separating steps can comprise the host cell of hGH, the tenuigenin of host cell or the cellular lysate of other materials, extract, substratum, inclusion body, carry out on the periplasmic space, or on any hGH mixture that obtains by any separating step, carry out, separating step includes, but is not limited to following steps: affinity chromatography, ion-exchange chromatography, the hydrophobic interaction chromatography, gel filtration chromatography, high performance liquid chromatography (" HPLC "), anti-phase HPLC (" RP-HPLC "), expanded bed adsorption or its any combination and/or repetition and with any suitable order.
Employed equipment and other necessary materials are commercially available in carrying out the technology of describing herein.Pump, run tank, monitor, register and whole system can be available from (for example) Applied Biosystems (Foster City, CA), Bio-Rad Laboratories, Inc. (Hercules CA) and GE Healthcare, and Inc. (Piscataway, NJ).The chromatographic material that includes, but is not limited to exchange group material, substratum and damping fluid also can be available from described company.
Such as balance and other steps in the column chromatography method of describing herein of washing and elution, can use such as the specific equipment of pump and finish more quickly.Commercially available pump includes, but is not limited to
Figure A20058004446300921
Pump P-50, peristaltic pump P-1, pump P-901 and pump P-903 (GE Healthcare, Piscataway, NJ).
The example of run tank comprise RediFrac run tank, FRAC-100 and FRAC-200 run tank and
Figure A20058004446300922
Run tank (GE Healthcare, Piscataway, NJ).Mixing tank also can be in order to form pH and linear concentration gradient.Commercially available mixing tank comprise mixing tank on gradient mixer GM-1 and the line (In-Line Mixer) (GE Healthcare, Piscataway, NJ).
Chromatographic process can use any commercially available monitor to monitor.Described monitor can be in order to collect the information as UV, pH value and specific conductivity.The example of detector comprise monitor UV-1,
Figure A20058004446300923
S II, monitor UV-M II, monitor UV-900, monitor UPC-900, monitor pH/C-900 and monitored conductivity device (GE Healthcare, Piscataway, NJ).In fact, comprise (Piscataway, NJ) various from GE Healthcare
Figure A20058004446300924
Whole systems of system are commercially available.
By described herein, the pH value of a hGH mixture can be regulated before carrying out any later separation step.In addition, under a hGH mixture or its any subsequent mixtures can be used in the field known technology concentrate.In addition, can use to the technology known to the those skilled in the art elution damping fluid that comprises a hGH mixture or its any subsequent mixtures is changed into the damping fluid that is suitable for next separating step.
Ion-exchange chromatography
In one embodiment and as optional, extra step, ion-exchange chromatography can be carried out on a hGH mixture.Usually referring to ION EXCHANGE CHROMATOGRAPHY:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1114-21, GE Healthcare (Piscataway, NJ)).Commercially available ion exchange column comprises
Figure A20058004446300931
Figure A20058004446300932
With
Figure A20058004446300933
Post (GE Healthcare, Piscataway, NJ).Described tubing string utilization is such as Q
Figure A20058004446300934
Fast Flow, Q
Figure A20058004446300935
High Performance and Q
Figure A20058004446300936
The strong anion exchanger of XL; Such as SP
Figure A20058004446300937
High Performance, SP
Figure A20058004446300938
Fast Flow and SP
Figure A20058004446300939
The strong cation exchanger of XL; Such as DEAE
Figure A200580044463009310
The weak anion exchanger of Fast Flow; With such as CM
Figure A200580044463009311
The weak cation exchanger of Fast Flow (GE Healthcare, Piscataway, NJ).Negatively charged ion or cation exchange column chromatography method can be carried out on hGH in any stage of purification process, to separate the hGH of purifying substantially.Source 30Q and Source 30S are ion exchange medium (GEHealthcare).
The cation-exchange chromatography step can use any suitable cationic exchange matrix to carry out.Effectively cationic exchange matrix includes, but is not limited to, fiber, porous, cationic exchange substrate material atresia, micromeritic, pearl or crosslinked.Described cationic exchange substrate material includes, but is not limited to, the matrix material of Mierocrystalline cellulose, agarose, dextran, polyacrylic ester, polyethylene, polystyrene, silica, polyethers or any previous materials.
Cationic exchange matrix can be any suitable cationite that comprises strong cation exchanger and weak cation exchanger.It is Ionized and therefore that strong cation exchanger can be left in wide pH value scope, can be in conjunction with hGH in wide pH value scope.Yet weak cation exchanger can lose ionization with the variation of pH value.For example, when pH is reduced to about pH 4 or pH 5 when following, weak cation exchanger can lose electric charge.The strong cation exchanger that is fit to includes, but is not limited to the charged functional groups such as sulfopropyl (SP), methylmesylate (S) or sulfoethyl (SE).Cationic exchange matrix can be to have about 2.5 to about 6.0 the hGH strong cation exchanger in conjunction with pH value scope.Perhaps, strong cation exchanger can have about pH 2.5 to the hGH of about pH 5.5 in conjunction with pH value scope.Cationic exchange matrix can be to have about 3.0 the hGH strong cation exchanger in conjunction with the pH value.Perhaps, cationic exchange matrix can be to have about 6.0 to about 8.0 the hGH strong cation exchanger in conjunction with pH value scope.Cationic exchange matrix can be to have about 8.0 to about 12.5 the hGH strong cation exchanger in conjunction with pH value scope.Perhaps, strong cation exchanger can have about pH 8.0 to the hGH of about pH 12.0 in conjunction with pH value scope.
Load before the hGH, cationic exchange matrix can (for example) be used the weak acid of the dilution of some column volumes, and for example the 20mM acetate of the pH 3 of 4 column volumes comes balance.After the balance, can add hGH and substantially before the hGH of purifying, use weakly acid soln again such as weak acetate or phosphoric acid solution in elution, with tubing string washing 1 to several times.For example, roughly the 20mM acetate of the pH 3 of 2-4 column volume can be in order to the sluicing pipe post.Also can use, use the 0.05M sodium acetate of the pH 5.5 of (for example) 2-4 column volume, or with the extra washing of the 0.1M sodium-chlor blended 0.05M sodium acetate of pH 5.5.Perhaps, known method in the field under using, cationic exchange matrix can use the weak base of the dilution of some column volumes to come balance.
Perhaps, the hGH of purifying can contact elution so that hGH is replaced from matrix with the damping fluid with enough low pH value or ionic strength by making cationite matrix substantially.The pH value of elution damping fluid can be at about pH 2.5 in the scope of about pH 6.0.More particularly, the pH value of elution damping fluid can be at about pH 2.5 to about pH 5.5, about pH 2.5 in the scope of about pH 5.0.The elution damping fluid can have about 3.0 pH value.In addition, the amount of elution damping fluid can change widely and will arrive in the scope of about 10 column volumes about 2 usually.In addition, can use herein, include, but is not limited to the damping fluid that is fit to known to the those skilled in the art, concentration at least about 5mM at least about the Citrate trianion in the scope of 100mM, phosphoric acid salt, formate, HEPES and MES damping fluid.
After hGH was adsorbed in cationite matrix, the hGH of purifying can contact elution so that hGH is replaced from matrix with the damping fluid with enough high pH value or ionic strength by making matrix substantially.The pH value of elution damping fluid can be at about pH 8.0 in the scope of about pH 12.5.More particularly, the elution damping fluid can be at about pH 8.0 in the scope of about pH 12.0.Be applicable to the elution of high pH value substantially the damping fluid that is fit to of the hGH of purifying include, but is not limited to, concentration at least about 5mM at least about the Citrate trianion in the scope of 100mM, phosphoric acid salt, formate, acetate, HEPES and MES damping fluid.In addition, can use the damping fluid of pH 8.7 with 0.1M potassium borate, 0.6M Repone K, 0.1mM EDTA.The hGH of purifying also can use standard buffer solution substantially, such as comprise pH 7.5 about 50 to the bicine of 100mM, the bicine of about 75mM; 25 arrive the sodium-chlor of about 100mM, are in particular the sodium-chlor of about 50mM; With about 0.05 to about 0.5 EDTA, more particularly the bicine damping fluid of the EDTA of about 0.1mM comes elution.
The anti-phase chromatography
Can carry out RP-HPLC and come protein purification according to being the scheme that is fit to known to the those skilled in the art.Referring to (for example), people such as Pearson, ANAL BlOCHEM. (1982) 124:217-230 (1982); People such as Rivier, J.CHROM. (1983) 268:112-119; People such as Kunitani, J.CHROM. (1986) 359:391-402.Can on hGH, carry out RP-HPLC and separate the hGH of purifying substantially.In this, can use alkyl functional base with multiple length through silica deutero-resin, include, but is not limited at least about C 3Arrive at least about C 30, at least about C 3Arrive at least about C 20, or at least about C 3Arrive at least about C 18Resin.Perhaps, can use polymer resin.For example, can use TosoHaas Amberchrome CG1000sd resin as styrenic polymer resins.Also can use cyano group or polymer resin with multiple alkyl chain length.In addition, the RP-HPLC post can be used such as the alcoholic acid solvent wash.Source RP post is another example of RP-HPLC post.
Contain ion-pairing agent and can be in order to from RP-HPLC post elution hGH such as the suitable elution damping fluid of methyl alcohol, Virahol, tetrahydrofuran (THF), acetonitrile or alcoholic acid organic modifiers.The most frequently used ion-pairing agent includes, but is not limited to, acetate, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, hyptafluorobutyric acid, triethylamine, tetramethylammonium, tetrabutylammonium, second triethylenetetraminehexaacetic acid ammonium.Can use one or more gradients or equality condition to carry out elution, wherein gradient condition is through preferably to reduce disengaging time and to reduce peak width.Usually, gradient can be in water about 5% to about 80% (v/v), about 5% to about 75% (v/v), about 5% to about 70% (v/v), about 5% to about 65% (v/v), about 5% to about 60% (v/v), about 5% to about 55% (v/v) or about 10% to about 50% (v/v) solvent.Another kind method relates to the use of two kinds of gradients with different solvents concentration range.The example that is applicable to elution damping fluid herein can include, but is not limited to ammonium acetate and acetonitrile solution.
The hGH that is obtained by the recombination bacillus coli host can pass through anti-phase chromatography isolated or purified in addition.Can (for example) use and have about 10% SOURCE RP post and separate hGH to the acetonitrile gradient of about 60% acetonitrile.
Hydrophobic interaction chromatography purification technology
Can on hGH, carry out hydrophobic interaction chromatography (HIC).Usually referring to, (catalog number (Cat.No.) 18-1020-90, (Piscataway, NJ), it is to incorporate this paper by reference into to GE Healthcare to HYDROPHOBICINTERACTION CHROMATOGRAPHY HANDBOOK:PRINCIPLES AND METHODS.The matrix that the HIC matrix that is fit to can (but being not limited to) replaces through alkyl or aryl, such as the matrix that replaces through butyl, hexyl, octyl group or phenyl, comprise agarose, Sepharose, sepharose, Mierocrystalline cellulose, silica, dextran, polystyrene, poly-(methacrylic ester) matrix and include, but is not limited to the polyvinylamine resin or the hybrid resin of poly-(methacrylic ester) matrix of replacing through butyl or phenyl.The commercially available source that is used for the hydrophobic interaction column chromatography includes, but is not limited to
Figure A20058004446300951
Figure A20058004446300952
With
Figure A20058004446300953
(GE Healthcare, Piscataway is NJ) with TSKgelPhenyl-650S and Phenyl-5PW (30um) resin (Tosoh Bioscience) for post.
In simple terms, before loading, the HIC tubing string can use and be the standard buffer solution known to the those skilled in the art, such as acetate/sodium chloride solution or contain the HEPES of ammonium sulfate, or the ammonium sulfate in the sodium radio-phosphate,P-32 solution of pH 6.5, or the sodium sulfate in the TRIS of pH 7-8 solution comes balance.Ammonium sulfate can be as the damping fluid that loads the HIC tubing string.After loading hGH, can use standard buffer solution subsequently and under the condition of all described conditions as described in this article, wash tubing string, to remove unwanted material but hGH is retained on the HIC tubing string.Can be with about 3 to the standard buffer solutions of about 10 column volumes, especially such as containing EDTA and than the HEPES damping fluid of level pad lower concentration ammonium sulfate, or acetate/sodium-chlor damping fluid comes elution hGH.The linear salt gradient that successively decreases of the gradient of use (for example) potassiumphosphate also can be in order to elution hGH molecule.Also can add the elution toughener that includes, but is not limited to ethylene glycol, glycerine or urea (0.5-1.5M) to the elution damping fluid.Then, (for example) concentrates eluant by the filteration such as diafiltration or ultrafiltration.Can utilize diafiltration to remove salt in order to elution hGH.
Other purification techniques
Use (for example) gel-filtration (GEL FILTRATION:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1022-18, GE Healthcare, Piscataway, NJ), it is to incorporate this paper by reference into, (suitable matrix includes, but is not limited to HA-Ultrogel to the hydroxyapatite chromatography method, High Resolution (Calbiochem), CHT pottery hydroxylapatite (BioRad), Bio-Gel HTP hydroxylapatite (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, diafiltration, lyophilize, suchlike another separating step can be carried out on a hGH mixture or its any subsequent mixtures, removing any excessive salt and with the damping fluid displacement damping fluid that is fit to, to be used for next separating step or even the prescription of final medicament production.
The non-naturally encoded amino acid that is present in the hGH molecule also can be in order to provide the separation that never contains non-naturally encoded amino acid whose other cell proteins.Because non-naturally encoded amino acid can comprise unique chemical functional group, so the coupling of unique functional group and another molecule can provide purification step substantially.For example, non-naturally encoded amino acid can with another molecule coupling that promotes from other protein separation.Be used for including, but is not limited to PEG and other polymkeric substance with the described molecule of non-natural amino acid coupling.
Can use to be the technology known to the those skilled in the art, each step monitoring of describing in this article comprises the productive rate of the hGH of the hGH of purifying substantially.Described technology also can be in order to assess behind the last separating step productive rate of the hGH of purifying substantially.For example, can use any tubing string in the some anti-phase high pressure liquid chromatography posts with various alkyl chain length, such as using cyano group RP-HPLC, C 18RP-HPLC; And the productive rate of cationic exchange HPLC and gel-filtration HPLC monitoring hGH.
In certain embodiments of the invention, the productive rate of the hGH behind each purification step can be in the initial substance of each purification step hGH at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9% or at least about 99.99%.
Purity can be used such as the standard technique of SDS-PAGE or by using west ink dot and ELISA calibrating measuring h GH to measure.For example, can produce the polyclonal antibody opposing and reclaim isolating protein from negativity contrast yeast fermentation and cationic exchange.Antibody also can be used to survey the existence of dyeing host cell proteins matter.
RP-HPLC material Vydac C4 (Vydac) is made up of silica gel particle, and its surface has the C4-alkyl chain.Make polypeptide separate the difference that is based on hydrophobic interaction intensity with protein impurities.Elution is to use the acetonitrile gradient in rare trifluoroacetic acid to carry out.Preparation property HPLC is to use stainless steel column (being filled with 2.8 to 3.2 liters Vydac C4 silica gel) to carry out.Hydroxylapatite Ultrogel eluant is acidifying and being loaded on the Vydac C4 post by adding trifluoroacetic acid.For washing and elution, use the acetonitrile gradient in rare trifluoroacetic acid.Collect cut and neutralize with phosphate buffered saline buffer immediately.Collect in the polypeptide cut in the IPC limit.
DEAE sepharose (GE Healthcare) material is made up of the diethyllaminoethyl that covalently is incorporated into the sepharose surface of beads (DEAE).Selected polypeptide is to mediate by ionic interaction with combining of DEAE group.Acetonitrile and trifluoroacetic acid do not keep by tubing string.After described material is washed out, remove trace impurity by acetate buffer washing tubing string with low pH value.Then, with neutral phosphonic phthalate buffer washing tubing string and with the damping fluid elution polypeptide of ionic strength with increase.Tubing string is filled up by the quick stream of DEAE sepharose.The adjustable column volume is to guarantee the polypeptide filler in the scope of 3-10mg polypeptide/ml gel.Water and level pad (sodium phosphate/potassiumphosphate) washing tubing string.Load the cut through compiling of HPLC eluant and wash tubing string with level pad.Then, with lavation buffer solution (sodium acetate buffer) washing, then wash with level pad.Subsequently, with elution damping fluid (sodium-chlor, sodium phosphate/potassiumphosphate) from tubing string elution polypeptide and according to the elution overview of standard with single fraction collection.The eluant of DEAE sepharose tubing string is adjusted to the specific conductivity of regulation.Make the medicine of gained be aseptically filled in the teflon bottle (Teflon bottle) and storage under-70 ℃.
Can comprise (but being not limited to) in order to the assessment productive rate of hGH and the method and the program of purity, Bradford calibrating, SDS-PAGE, silver dyeing SDS-PAGE, Xylene Brilliant Cyanine G (coomassie) dyeing SDS-PAGE, mass spectrometry (including, but is not limited to MALDI-TOF) and be the additive method that is used for profiling protein matter known to the those skilled in the art.Extra method includes, but is not limited to: with SDS-PAGE, immune ink dot method (immunoblotting), substance assistant laser desorpted pair of effect/ionization-mass spectrometry (MALDI-MS), liquid chromatography/mass spectrometry, isoelectrofocusing, analytical anionresin, chromatofocusing and the circular dichroism of protein staining method coupling.
Operable additional method comprises removes endotoxic step.Intracellular toxin is the lipopolysaccharides material that is positioned at such as on the outer membrane of the Gram-negative host cell of colon bacillus.The method that reduces level of endotoxin is for known to the those skilled in the art and include, but is not limited to use the suchlike purification technique of combination of silica upholder, glass powder or hydroxylapatite, anti-phase chromatogram, avidity chromatogram, size exclusion chromatography, anion-exchange chromatography, hydrophobic interaction chromatography, filtration, described method.Can need to revise or extra method is removed such as the proteinic pollutent from the common migration of the polypeptide of being paid close attention to.The method of measuring level of endotoxin is for known to the those skilled in the art and include, but is not limited to king crab amebocyte lysate (Limulus Amebocyte Lysate) and (LAL) examine and determine.
Although describe the present invention, it will be apparent to those skilled in the art that and to carry out various changes and modification without departing from the invention with reference to specific embodiment, method, construction and purposes.It is conspicuous that indicated reagent, material and purification condition are changed the those skilled in the art.For example, more the resin of heavy body if described capacity is desired, can substitute in the chromatography step so.
XIV. the expression in the alternative system
Used some strategies with at the non-recombinant hosts cell, in the host cell of sudden change or in cell free system, alpha-non-natural amino acid has been introduced in the protein.Described system also is applicable to and makes hGH polypeptide of the present invention.The amino acid that derivatize has reactive side chain such as Lys, Cys and Tyr can cause Methionin to N 2The conversion of-ethanoyl-Methionin.Chemosynthesis also provides the direct method of incorporating alpha-non-natural amino acid into.Along with the enzymatic of peptide fragment connects the immediate development that is connected with native chemical, might make bigger protein.Referring to (for example), P.E.Dawson and S.B.H.Kent, Annu.Rev.Biochem,69:923 (2000).The chemistry peptide connects to be connected with native chemical and is described in United States Patent (USP) the 6th, 184, among No. 344, No. the 2004/0138412nd, U.S. Patent Publication case, No. the 2003/0208046th, U.S. Patent Publication case, WO 02/098902 and the WO 03/042235, described patent is to incorporate this paper by reference into.With the inhibition tRNA through desired alpha-non-natural amino acid chemical acylation add to can the biosynthetic in vitro extract of nebulin matter generally biosynthetic means in vitro, in order to incorporating into specifically in the range protein with any physical size above the alpha-non-natural amino acid position more than 100 kinds.Referring to (for example), V.W.Cornish, D.Mendel and P.G.Schultz, Angew.Chem.Int.Ed.Engl.,1995,34:621 (1995); C.J.Noren, S.J.Anthony-Cahill, M.C.Griffith, P.G.Schultz, A general method for site-specific incorporation of unnatural aminoacids into proteins, Science244:182-188 (1989); With, J.D.Bain, C.G.Glabe, T.A.Dix, A.R.Chamberlin, E.S.Diala, Biosynthetic site-specific incorporation of a non-natural amino acidinto a polypeptide, J.Am.Chem.Soc.111:8013-8014 (1989).The functional group with broad range introduces in the protein to be used to study protein stability, protein folding, enzyme mechanism and signal transduction.
Exploitation is called in vivo method that selection pressure incorporates into to utilize the scrambling of wild-type synthetic enzyme.Referring to (for example), N.Budisa, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L.Moroder and R.Huber, FASEB J., 13:41 (1999).The cut auxotrophy bacterial strain in associated metabolic path of supplying with the concrete natural amino acid of cell is to grow in the minimal medium of the natural amino acid that contains limited concentration, and transcribing of target gene is suppressed.When stationary growth phase began, natural amino acid was depleted and replace through the alpha-non-natural amino acid analogue.Induce the expression of recombinant protein to cause the proteinic accumulation that contains the non-natural analogue.For example, use described strategy, ortho position, a position and contraposition fluorophenylalanine have been merged in the protein and have shown two feature shoulder shape projections in the UV spectrum that can be identified easily, referring to (for example), C.Minks, R.Huber, L.Moroder and N.Budisa Anal. Biochem., 284:29 (2000); The fluoroform methyllanthionine in order to the methionine(Met) in the displacement phage T4 N,O-Diacetylmuramidase to pass through 19F NMR studies the interaction of itself and oligochitosan ligand, referring to (for example), and H.Duewel, E.Daub, V.Robinson and J.F.Honek, Biochemistry,36:3404 (1997); And the trifluoro L-LEU has been merged in the replacement L-LEU, causes the thermostability of L-LEU-zipper protein white matter and chemical stability to increase.Referring to (for example), Y.Tang, G.Ghirlanda, W.A.Petka, T.Nakajima, W.F.DeGrado and D.A.Tirrell, Angew.Chem.Int. Ed.Engl., 40:1494 (2001).In addition, selenomethionine and telluro methionine(Met) are merged in the various recombinant proteins promoting in X-ray crystallography and separate mutually.Referring to (for example), W.A.Hendrickson, J.R.Horton and D.M.Lemaster, EMBO J.,9:1665 (1990); J.O.Boles, K.Lewinski, M.Kunkle, J.D.Odom, B.Dunlap, L.Lebioda and M.Hatada, Nat.Struct.Biol..1:283 (1994); N.Budisa, B.Steipe, P.Demange, C.Eckerskorn, J.Kellermann and R.Huber, Eur.J.Biochem., 230:788 (1995); And N.Budisa, W.Karnbrock, S.Steinbacher, A.Humm, L.Prade, T.Neuefeind, L.Moroder and R.Huber, J.Mol.Biol.,270:616 (1997).Methionine(Met) analogue with alkene or alkynes functional group is also incorporated into effectively, allows by the extra modifying protein of chemical means.Referring to (for example), J.C.van Hest and D.A.Tirrell, FEBS Lett., 428:68 (1998); J.C.van Hest, K.L.Kiick and D.A.Tirrell, J.Am.Chem. Soc.122:1282 (2000); With K.L.Kiick and D.A.Tirrell, Tetrahedron, 56:9487 (2000); United States Patent (USP) the 6th, 586, No. 207; U.S. Patent Publication case 2002/0042097, it is to incorporate this paper by reference into.
The success of described method depends on by aminoacyl-tRNA synthetase and distinguishes the alpha-non-natural amino acid analogue that generally speaking, it needs the high fidelity of reproduction of selectivity to guarantee that protein is translated.A kind of mode of expanding the category of described method is to relax the substrate specificity of aminoacyl-tRNA synthetase, and it is reached under condition of limited.For example, by in colon bacillus phenylalanyl-tRNA synthetic enzyme (PheRS), replacing Ala with Gly 294Can increase the size of substrate binding pocket, and cause fenclonine (p-Cl-Phe) acidylate tRNAPhe.Referring to, M.Ibba, P.Kastand H.Hennecke, Biochemistry, 33:7107 (1994).The colon bacillus bacterial strain of nourishing described mutant PheRS is allowed and is incorporated fenclonine into or bromophenyl alanine is replaced phenylalanine.Referring to (for example), M.Ibba and H.Hennecke, FEBS Lett..364:272 (1995); With, N.Sharma, R.Furter, P.Kast and D.A.Tirrell, FEBS Lett..467:37 (2000).Similarly, the point mutation Phel30Ser that has showed the amino acid combining site of close colon bacillus tyrosyl-t RNA synthetase allows that azatyrosine more effectively incorporates into than tyrosine.Referring to, F.Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soll and S.Nishimura, J.Biol.Chem.,275:40324 (2000).
Alpha-non-natural amino acid being incorporated into proteinic another strategy in vivo is to modify the synthetic enzyme with check and correction mechanism.Described synthetic enzyme can not be distinguished on the structure similar in appearance to the amino acid of homology natural amino acid and therefore can not activate described amino acid.Described error is corrected at separated part, and described separated part will go acidylate to keep the fidelity of reproduction that protein is translated from the amino acid through mistake filling (mischarged) of tRNA.If the check and correction activity of forfeiture synthetic enzyme can be avoided editting function and is merged in through wrong activatory analog so.Used valyl-tRNA synthetic enzyme (ValRS) to prove described method in the recent period.Referring to, V.Doring, H.D.Mootz, L.A.Nangle, T.L.Hendrickson, V.de Crecy-Lagard, P.Schimmel and P.Marliere, Science, 292:501 (2001).ValRS can use Cys, Thr or aminobutyric acid salt (Abu) with the amino acidylate of tRNAVal mistake; Then, make described non-homogeneous amino acid hydrolysis by edit field.Behind the chromosomal random mutation of colon bacillus, the editor position that is chosen in ValRS has the sudden change colon bacillus bacterial strain of sudden change.The described editor's of lacking ValRS loads tRNAVal with Cys improperly.Because be similar on the Abu space Cys (Cys-the SH group in Abu-CH3 displacement), so when described sudden change colon bacillus bacterial strain is grown in the presence of Abu, the ValRS that suddenlys change also incorporates Abu in the protein into.Mass spectroscopy shows that in primary protein, about 24% Xie Ansuan is replaced through Abu in each Xie Ansuan position.
Solid phase synthesis and semisynthesis also allow to contain a large amount of proteinic synthetic of new amino acid.For example, referring to following discloses case and the reference wherein quoted, described open case is as follows: Crick, F.H.C., Barrett, L.Brenner, S.Watts-Tobin, R.General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H.Studies on polypeptides.XXXVI.The effect ofpyrazole-imidazole replacements on the S-protein activating potency of an S-peptidefragment, J.Am Chem, 88 (24): 5914-5919 (1966); Kaiser, E.T.Synthetic approaches tobiologically active peptides and proteins including enyzmes, Ace Chem Res, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E.T.Peptide segment coupling catalyzed by thesemisynthetic enzyme thiosubtilisin, J Am Chem Soc.109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256 (5054): 221-225 (1992); Chaiken, I.M.Semisynthetic peptides and proteins, CRC Crit Rev Biochem.11 (3): 255-301 (1981); Offord, R.E.Protein engineering by chemical means? Protein Eng..1 (3): 151-157 (1987); And Jackson, D.Y., Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, J.A.A Designed Peptide Ligase forTotal Synthesis of Ribonuclease A with Unnatural Catalytic Residues, Science, 266 (5183): 243 (1994).
Chemically modified is in vitro introduced in the protein in order to the various non-natural side chains that will comprise cofactor, spin label and oligonucleotide.Referring to (for example), Corey, D.R., Schultz, P.G.Generation of a hybridsequence-specific single-stranded deoxyribonuclease, Science, 238 (4832): 1401-1403 (1987); Kaiser, E.T., Lawrence D.S., Rokita, S.E.The chemical modification of enzymaticspecificity, Annu Rev Biochem, 54:565-595 (1985); Kaiser, E.T., Lawrence, D.S.Chemicalmutation of enyzme active sites, Science, 226 (4674): 505-511 (1984); Neet, K.E., Nanci A, Koshland, D.E.Properties of thiol-subtilisin, J Biol.Chem,243 (24): 6392-6401 (1968); Polgar, L.et M.L.Bender.A new enzyme containing a synthetically formed active site.Thiol-subtilisin. J.Am Chem Soc,88:3153-3154 (1966); And Pollack, S.J., Nakayama, G.Schultz, P.G.Introduction of nucleophiles and spectroscopic probes into antibody combiningsites, Science,242 (4881): 1038-1040 (1988).
Perhaps, use biosynthetic means through the aminoacyl-tRNA of chemically modified in order to some biophysics probes are in vitro incorporated in institute's synthetic protein.Referring to following discloses case and the reference wherein quoted: Brunner, J.New Photolabeling and crosslinking methods, Annu.Rev Biochem,62:483-514 (1993); With, Krieg, U.C., Walter, P., Hohnson, A.E.Photocrosslinking of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the signal recognition particle Proc.Natl. Acad.Sci, 83 (22): 8604-8608 (1986).
Showed in the past, and can react by the protein synthesis that the inhibition tRNA through the amino acidylate of chemistry is added to the gene programization that contains desired amber nonsense mutation, and in vitro alpha-non-natural amino acid site specific ground is incorporated in the protein.Use described method, the those skilled in the art can replace a large amount of 20 common seed amino acids with the homologue of enclosed construction, and for example, fluorophenylalanine substituted benzene L-Ala uses the auxotrophy bacterial strain to replace concrete amino acid.Referring to (for example), Noren, C.J., Anthony-Cahill, Griffith, M.C., Schultz, P.G.A general methodfor site-specific incorporation of unnatural amino acids into proteins, Science,244:182-188 (1989); M.W.Nowak waits the people, Science268:439-42 (1995); Bain, J.D., Glabe, C.G., Dix, T.A., Chamberlin, A.R., Diala, E.S.Biosynthetic site-specific Incorporation of a non-naturalamino acid into a polypeptide, J.Am Chem Soc,111:8013-8014 (1989); People such as N.Budisa, FASEB J.13:41-51 (1999); Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G.Biosynthetic method for introducing unnatural amino acids site-specifically intoproteins. Methods in Enz., vol.202,301-336 (1992); And Mendel, D., Cornish, V.W.﹠amp; Schultz, P.G.Site-Directed Mutagenesis with an Expanded Genetic Code, Annu Rev Biophys.Biomol Struct.24,435-62 (1995).
For example, the preparation distinguish the inhibition tRNA of terminator codon UAG and with alpha-non-natural amino acid with its chemical amino acidylate.Habitual positional mutation is introduced terminator codon TAG in order to the position of being paid close attention in protein gene.Referring to (for example), Sayers, J.R., Schmidt, W.Eckstein, F.5 '-3 ' Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, Nucleic Acids Res, 16 (3): 791-802 (1988).When the inhibition tRNA of acidylate and mutator gene in vitro make up in the transcription/translation system, incorporate alpha-non-natural amino acid into codon with response UAG, obtain containing that amino acid whose protein at prescribed position.Use [ 3H]-experiment of Phe and incorporate into by the UAG codon in the position of regulation with the only desired amino acid of experiment showed, of α-carboxylic acid, and described amino acid is not incorporated at the position of any other in protein.Referring to (for example), Noren waits the people, and is the same; People such as Kobayashi, (2003) Nature Structural Biology 10 (6): 425-432; And Ellman, J.A., Mendel, D., Schultz, P.G.Site-specific incorporation of novel backbonestructures into proteins, Science, 255 (5041): 197-200 (1992).
Can be by including, but is not limited to any method or the technology of the amino acidylate of chemical amino acidylate or enzymatic, with desired amino amino acidylate tRNA.
Amino acidylate can be finished by aminoacyl tRNA synthetase or by other enzymatic molecules that include, but is not limited to ribozyme.Term " ribozyme " can exchange with " catalytic RNA ".Cech and colleague (Cech, 1987, Science, 236:1532-1539; People such as McCorkle, 1987, Concepts Biochem.64:221-226) proved the existence of the RNA of the natural generation that can serve as catalyzer (ribozyme).Yet, only the Yeast Nucleic Acid substrate that is used for cracking and montage to be worked although showed described natural RNA catalyzer, the immediate development of the artificial evolution of ribozyme expands to various chemical reactions with the inventory of katalysis.Research identified can himself (2 ') 3 '-end on the RNA molecule (people such as Illangakekare of catalytic amino acyl-RNA key, 1995 Science 267:643-647) and can be with amino acid (the people such as Lohse of the RNA molecule from a RNA molecular transfer to another, 1996, Nature 381:442-444).
The U.S. Patent Application Publication case 2003/0228593 of incorporating this paper by reference into is described the method for construction ribozyme and its with the purposes among the amino amino acidylate tRNA natural coding and non-naturally encoded.Include, but is not limited to ribozyme can amino acidylate tRNA the substrate consolidated form of enzymatic molecule, can facilitate effective avidity purifying through the product of amino acidylate.The example of the substrate that is fit to comprises agarose, sepharose and magnetic beads.The machine made production of substrate and the purposes that are used for the ribozyme of amino acidylate are described in Chemistry and Biology 2003, and in 10:1077-1084 and the U.S. Patent Application Publication case 2003/0228593, it incorporates this paper by reference into.
The amino process for acylating of chemistry includes, but is not limited to, by Hecht and colleague (Hecht, S.M.Ace.Chem.Res.1992,25,545; Heckler, T.G.; Roesser, J.R.; Xu, C; Chang, P.; Hecht, S.M.Biochemistry1988,27,7254; Hecht, S.M.; Alford, B.L.; Kuroda, Y.; Kitano, S.J.Biol.Chem.1978,253,4517) and by Schultz, Chamberlin, Dougherty and other people (Cornish, V.W.; Mendel, D.; Schultz, P.G.Angew.Chem.Int.Ed.Engl.1995,34,621; Robertson, S.A.; Ellman, J.A.; Schultz, P.G.J.Am.Chem.Soc.1991,113,2722; Noren, C.J.; Anthony-Cahill, S.J.; Griffith, M.C; Schultz, P.G.Science 1989,244, and 182; Bain, J.D.; Glabe, C.G.; Dix, T.A.; Chamberlin, A.R.J.Am.Chem.Soc.1989,111,8013; Bain, people Nature such as J.D. 1992,356,537; Gallivan, J.P.; Lester, H.A.; Dougherty, D.A.Chem.Biol.1997,4,740; People J.Biol.Chem.1996 such as Turcatti, 271,19991; Nowak, people Science such as M.W., 1995,268,439; Saks, people J.Biol.Chem.1996 such as M.E., 271,23169; Hohsaka, people J.Am.Chem.Soc.1999 such as T., 121,34) the described method of avoiding in amino acylation, using synthetic enzyme that proposes, it incorporates this paper by reference into.Described method or other chemical amino process for acylating can be in order to aminoacyl tRNA molecules.
The method that is used to produce catalytic RNA can relate to the separate tank that produces randomized ribozyme sequence, carries out orthogenesis on the pond, screen the pond and select to show the sequence of the described ribozyme of desired aminoacylation activity for required aminoacylation activity.
Ribozyme can include and is beneficial to active primitive of acidylate and/or zone, such as GGU primitive and U rich region.For example, reported that the U rich region can promote distinguishing of amino acid substrate, and the GGU-primitive can form base pair with 3 of tRNA ' end.In when combination, distinguish when GGU and primitive and U rich region promote amino acid and tRNA simultaneously, and and then promote the amino acidylate that 3 of tRNA ' is terminal.
Ribozyme can be by using and tRNA AsnThe r24mini of CCCG bonded incomplete randomization in vitro selects, and then produces by the systems engineeringization that sees the concensus sequence in the active clone.The exemplary ribozyme that obtains by described method is called as " Fx3 ribozyme " and is described in No. the 2003/0228593rd, the open application case of the U.S.; the content of described patent is to incorporate this paper by reference into, and described ribozyme is taken on the multi-usage catalyzer and is used for the synthetic various aminoacyl-tRNAs that load through the homology alpha-non-natural amino acid.
Fixing on the substrate can be in order to facilitate the effective avidity purifying through the tRNA of amino acidylate.The example of the substrate that is fit to includes, but is not limited to agarose, sepharose and magnetic beads.Ribozyme can be fixed in by the chemical structure of utilizing RNA on the resin, such as 3 on the ribose of RNA '-the cis glycol can be through periodate oxidation to produce corresponding dialdehyde to promote RNA fixing on resin.Can use the various types of resins that comprise cheap hydrazides resin, wherein reductive amination makes the interaction between resin and the ribozyme become irreversible key.The synthetic of aminoacyl-tRNA can promote significantly by amino acidylate technology on the described tubing string.People Methods 2005 such as Kourouklis; 36:239-4 describes the amino acidylate system based on tubing string.
Separation through the tRNA of amino acidylate can be finished in every way.A kind of suitable method is, from have damping fluid such as the sodium acetate solution that contains 10mM EDTA, contain 50mM N-(2-hydroxyethyl) piperazine-N '-(3-propane sulfonic acid), pH7.0 12.5mM KCl, 10mM EDTA damping fluid or only for through the tubing string elution of the water (pH7.0) of edta buffer tRNA through amino acidylate.
TRNA through amino acidylate can be added to and translate reaction, so that incorporate amino acid into, by translating on the privileged site in the polypeptide that makes of reaction, tRNA is by described amino amino acidylate.Can use the example of the system that translates of the tRNA through amino acidylate of the present invention, include, but is not limited to cellular lysate.Cellular lysate is provided as from input mRNA and in vitro translates the necessary reactive component of polypeptide.The example of described reactive component include, but is not limited to ribosomal protein, rRNA, amino acid, tRNA, GTP, ATP, the rotaring intertranslating start factor and elongation factor with translate other relevant factors.In addition, the system of translating translates in batches or separates to translate.The system that in batches translates composite reaction component in single compartment makes and translates reactive component and separate with the reaction product that can suppress to translate effect and separate the system of translating.The described system of translating is commercially available.
In addition, can use coupling transcription/translation system.Coupling transcription/translation system allows the DNA of input is transcribed into corresponding mRNA, and mRNA is translated by reactive component.The example of commercially available coupling transcription/translation is RapidTranslation System (RTS, Roche Inc.).System comprises containing to be useful on to be provided such as the ribosomal intestinal bacteria lysate of component and the mixture of translation factor of translating.In addition, comprise that RNA polymerase is used for input DNA is transcribed into and is applicable to the mRNA template of translating.RTS can use by the film between the insertion reaction compartment (comprising supply/consumption compartment and transcription/translation compartment) reactive component is separated.
The amino acidylate of tRNA can be by including, but is not limited to transferring enzyme, polysaccharase, catalytic antibody, multifunctional protein, and suchlike other reagent are carried out.
People such as Lu are at Mol Cell.2001 Oct; 8 (4): describe the method (being connected) that protein and the synthetic chemistry of peptides that contains alpha-non-natural amino acid are engaged among the 759-69 through expressed protein.
Microinjection technique also has been used for incorporating alpha-non-natural amino acid into protein.Referring to (for example), M.W.Nowak, P.C.Kearney, J.R.Sampson, M.E.Saks, C.G.Labarca, S.K.Silverman, W.G.Zhong, J.Thorson, J.N.Abelson, N.Davidson, P.G.Schultz, D.A.Dougherty and H.A.Lester Science.268:439 (1995); And D.A.Dougherty, Curr.Opin.Chem.Biol., 4:645 (2000).Xenopus leavis oocytes (Xenopus oocyte) is to inject jointly with following two kinds of RNA materials that in vitro make: be coded in the amino acid position of being paid close attention to have the UAG terminator codon target protein mRNA and through the amber suppressor tRNA of the amino acidylate of desired alpha-non-natural amino acid.Then, ovocyte translate machine the regulation the position on, by UAG alpha-non-natural amino acid is inserted.Described method has made can carry out the in vivo structure-functional study of inherent membranin, and described inherent membranin is disobeyed in vitro expression system usually.Example comprises incorporates in tachykinin neurokinin-2 acceptor fluorescence amino acid to transmit measuring distance by fluorescence resonance energy into, referring to (for example), G.Turcatti, K.Nemeth, M.D.Edgerton, U.Meseth, F.Talabot, M.Peitsch, J.Knowles, H.Vogel and A.Chollet J.Biol.Chem., 271:19991 (1996); Incorporate biotinylation amino acid into the surperficial exposed residue in the identification ionic channel, referring to (for example), J.P.Gallivan, H.A.Lester and D.A.Dougherty, Chem.Biol., 4:739 (1997); Use cage tyrosine analogue with the conformational change in the real-time monitoring ionic channel, referring to (for example), J.C.Miller, S.K.Silverman, P.M.England, D.A.Dougherty and H.A.Lester, Neuron, 20:619 (1998); Be used to survey its door control mechanism with use α hydroxy-amino-acid to change the ionic channel framework.Referring to (for example), P.M.England, Y.Zhang, D.A.Dougherty and H.A.Lester, Cell, 96:89 (1999); And T.Lu, A.Y.Ting, J.Mainland, L.Y.Jan, P.G.Schultz and J.Yang, Nat.Neurosci., 4:239 (2001).
The ability of directly alpha-non-natural amino acid in vivo being incorporated in the protein provides the multiple advantage that includes, but is not limited to following advantage: the high yield of mutein, technology simplification, may or may study mutein in cell and use described mutein in live organism in therapeutic treatment and diagnostic uses.Make have various size, the alpha-non-natural amino acid of acidity, nucleophilicity, hydrophobicity and other character the ability in the protein of being included in can expand our the reasonably and systematically ability of the structure of operon protein widely, to survey protein function and to produce novel protein or the organism with novel character.Yet,, reach the interactional complicated character of the needed tRNA-synthetic enzyme of fidelity of reproduction of high level, so described method is difficult because in protein is translated.
In once trial to F-Phe is incorporated on site specific ground into, that yeast amber suppressor tRNAPheCUA/ phenylalanyl-tRNA synthetic enzyme is anti-to F-Phe, Phe auxotroph colon bacillus bacterial strain to being used for.Referring to (for example) R.Furter, Protein Sci.,7:419 (1998).
Also may use acellular (in vitro) system of translating to obtain the expression of hGH polynucleotide of the present invention.The system of translating can be cell or acellular, and can be protokaryon or eucaryon.The cell system of translating includes, but is not limited to full cell preparation or the cell culture such as the permeability cell, wherein desired nucleotide sequence can be transcribed into mRNA and mRNA is translated.The acellular system of translating is that commercially available and many dissimilar and systems are known.The example of cell free system includes, but is not limited to, such as the protokaryon lysate of colon bacillus lysate with such as the eucaryon lysate of wheat malt germ extract, insect cell lysate, rabbit skein cell lysate, rabbit oocyte lysate and human cell's lysate.When gained protein through glycosylation, phosphorylation or otherwise when modified, eucaryon extract or lysate can be preferably, because many described modifications only are possible in eukaryotic system.Some described extracts and lysate are commercially available (Promega; Madison, Wis.; Stratagene; La Jolla, Calif; Amersham; Arlington Heights, Ill.; GIBCO/BRL; Grand Island, N.Y.).Membranous extract such as the dog pancreatic extract that contains microsomal membrane also is an available, and it is applicable to translates secreted protein.Can comprise mRNA as template (in vitro translating) or DNA as the described system of template (in vitro transcribing and translating) through combination in, in vitro synthetic is to guide by rrna.Exploitation cell-free protein expression system is carried out sizable effort.Referring to (for example), Kim, D.M. and J.R.Swartz, Biotechnology and Bioengineering, 74:309-316 (2001); Kim, D.M. and J.R.Swartz, Biotechnology Letters, 22,1537-1542, (2000); Kim, D.M. and J.R.Swartz, Biotechnology Progress, 16,385-390, (2000); Kim, D.M. and J.R.Swartz, Biotechnology and Bioengineering, 66,180-188, (1999); And Patnaik, R. and J.R.Swartz, Biotechniques 24,862-868, (1998); United States Patent (USP) the 6th, 337, No. 191; No. the 2002/0081660th, U.S. Patent Publication case; WO 00/55353; WO 90/05785, and it is to incorporate this paper by reference into.Can be applied to express the another kind of method that comprises non-naturally encoded amino acid whose hGH polypeptide and comprise mRNA-peptide integration technology.Referring to (for example), R.Roberts and J.Szostak, Proc.Natl Acad.Sci. (USA) 94:12297-12302 (1997); People such as A.Frankel, Chemistry ﹠amp; Biology 10:1043-1050 (2003).In described method, the mRNA template that is connected with tetracycline is translated into peptide on rrna.If modified one or more tRNA molecules so also can be incorporated non-natural amino acid in the peptide into.Behind once the mRNA codon, tetracycline is captured the C-end of peptide on reading out.Have the character of being paid close attention to if find gained mRNA-peptide binding substances in vitro in the calibrating, its identity can easily disclose from the mRNA sequence so.Therefore, the those skilled in the art can screen the storehouse that comprises one or more non-naturally encoded amino acid whose hGH polypeptide, the polypeptide that has desired character with identification.In the recent period, be reported that carrying out rrna in vitro with purified component translates and make and can synthesize peptide through non-naturally encoded aminoacid replacement.Referring to (for example), people such as A.Forster, Proc.Natl Acad.Sci. (USA) 100:6353 (2003).
Also can use reconstruct to translate system.The mixture of purified translation factor and also successful in order to mRNA is translated in the protein by combination or lysate such as the additional lysate of the purified translation factor of initiation factor-1 (IF-1), IF-2, IF-3 (α or β), EF-T (EF-Tu) or terminator factor.Cell free system also can be the transcription/translation system through coupling, wherein as Current Protocols in Molecular Biology (editor such as F.M.Ausubel, Wiley Interscience, 1993) described in the DNA drawing-in system, be transcribed into mRNA and translate mRNA, it is incorporated into especially by reference at this.The RNA that in the eukaryotic transcription system, transcribes can be heteronuclear RNA (hnRNA) or 5 '-distal end cap (7-methyl guanosine) and 3 '-art end poly A has the ripe mRNA form of tail, it can be some and translates advantage in the system.For example, adding cap mRNA is translated in skein cell lysate system with high-level efficiency.
XV. with the high polymer of hGH polypeptide coupling
The various modifications to non-natural amino acid polypeptide of Miao Shuing herein can use composition, method, technology and the strategy described to realize herein.Described modification comprises that another functional group who will include, but is not limited to following material incorporates on non-natural amino acid composition of polypeptide: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; The photocrosslinking agent; Radionuclide; Cytotoxin compounds; Medicine; Affinity labelling; Photoaffinity labeling; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotides; Carbohydrate; Water miscible branch-shape polymer; Cyclodextrin; Inhibition Yeast Nucleic Acid; Biomaterial; Nanoparticle; Spin label; Fluorophore, metallic part; Radioactive segment; Novel functional group; Covalently or non-covalentlyly with the group of other interactions of molecules; The photic part of shrouding; But the part that actinic radiation excites; But the part of photoisomerization; Vitamin H; The derivative of vitamin H; The vitamin H analogue; The part of incorporating heavy atom into; But the group of chemical cracking; But the group of photo-cleavage; The side chain of elongation; Sugar through the carbon connection; The redoxomorphism promoting agent; Amino thioic acid sulfoacid; The toxicity part; Isotope-labeled part; The biophysics probe; Phosphorescent group; Chemiluminescent group; The close group of electronics; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable mark; Small molecules; Quantum dot; The nanometer transmitter; The radioactive nuleus thuja acid; The radioactivity transmitter; Neutron capture agent; Or any combination of above-mentioned substance, or any other required compound or material.Illustrative, non-limiting instance as the composition of describing herein, method, technology and strategy, below describe and add high polymer to non-natural amino acid polypeptide concentrating on, condition is that composition, method, technology and the strategy wherein described (if necessary and those skilled in the art can carry out with disclosure herein the time, so its can through suitable modification) also is applicable to and adds other functional substance that include, but is not limited to described functional substance listed above.
Multiple high polymer and other molecules can be connected with hGH polypeptide of the present invention regulating the biological property of hGH polypeptide, and/or provide novel biological property for the hGH molecule.Described high polymer can be by natural amino acids coding, by the natural or non-natural amino acid whose any sense substituent of non-naturally encoded amino acid, or add natural or non-natural amino acid whose any substituting group or functional group to and be connected with the hGH polypeptide.The molecular weight of polymkeric substance can have and includes, but is not limited at about 100Da and about 100 000Da or 100, the wide region between 000Da is above.The molecular weight of polymkeric substance can between the 000Da, include, but is not limited to 100 at about 100Da and about 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.
The invention provides polymkeric substance: the preparation of the homogeneous substantially of protein conjugate.By used herein, " homogeneous substantially " meaning is to observe polymkeric substance: the protein conjugate molecule is greater than half of gross protein.Polymkeric substance: protein conjugate biologically active and the present invention of providing herein " homogeneous substantially " are to be enough homogeneous with the described preparation of the advantage that shows homogeneous preparation through the hGH of Pegylation polypeptide formulations, for example are easy to predict the pharmacokinetics of between-lot in clinical application.
The those skilled in the art also can select to prepare polymkeric substance: the mixture of protein conjugate molecule, and the advantage that provides herein is that the those skilled in the art can select single polymkeric substance: protein conjugate is included in the ratio in the mixture.Therefore, if desired, the those skilled in the art can prepare range protein and (that is to say with the polymer moieties of the various quantity that are connected so, dimerization, trimerization, four poly-etc.) mixture, and single polymkeric substance: protein conjugate combination, and the single polymkeric substance that obtains having predetermined proportion: the mixture of protein conjugate with described binding substances and use method preparation of the present invention.
Selected polymkeric substance can be water miscible so that connected protein does not precipitate in the aqueous environment such as physiological environment.Polymkeric substance can be ramose or ramose not.Concerning the therapeutic use of terminal point product preparation, polymkeric substance will be for pharmaceutically acceptable.
The example of polymkeric substance includes, but is not limited to poly alkyl ether and its alkoxyl group (for example adds the cap analogue, polyoxyethylene glycol (polyoxyethylene glycol), polyoxyethylene/propylene glycol, add the cap analogue with its methoxy or ethoxy, especially be polyoxyethylene glycol, the latter is also referred to as polyoxyethylene glycol or PEG); Polyvinylpyrrolidone; The polyvinyl alkyl oxide; Poly-oxazoline, poly-alkylated oxazoline quinoline and poly-hydroxyalkyl oxazoline; Polyacrylamide, poly-alkyl acrylamide and poly-hydroxyalkyl acrylamide (for example, poly-hydroxypropylmethyl acrylamide and its derivative); Poly-hydroxyalkyl acrylates; Polysialic acid and its analogue; The hydrophilic peptide sequence; Polysaccharide and its derivative of comprising dextran and glucan derivative, for example, Sensor Chip CM 5, dextran sulfate, glycosaminoglycan; Mierocrystalline cellulose and its derivative, for example, carboxymethyl cellulose, hydroxy alkyl cellulose; Chitin and its derivative, for example chitosan, succinyl chitosan, carboxymethyl chitosan, carboxymethyl chitosan; Hyaluronic acid and its derivative; Starch; Alginates; Chondroitin sulfate; Albumin; Amylopectin and carboxymethyl amylopectin; Polyamino acid and its derivative, for example, polyglutamic acid, polylysine, poly-aspartic-acid, poly-asparagine; Maleic anhydride copolymer, such as: styrene maleic anhydride copolymer, divinyl ethyl ether maleic anhydride copolymer; Polyvinyl alcohol; Its multipolymer; Its trimer; The derivative of its mixture and aforementioned polymer.
Its concentration will change the same in reaction mixture, and the ratio of peg molecule and protein molecule will change.Generally speaking, best ratio (with regard to the effect of the reaction that has minimum excessive unreacted protein or polymkeric substance) can be determined by the molecular weight of selected polyoxyethylene glycol and the quantity available of available reactive group.When relating to molecular weight, the molecular weight of polymkeric substance is high more usually, and the quantity of the polymer molecule that can be connected with protein is just few more.Similarly, when optimizing described parameter, should consider the branch of polymkeric substance.Usually, molecular weight high more (or branch is many more), polymkeric substance: protein rate is just high more.
By used herein and when containing the PEG:hGH polypeptide conjugates, term " treatment significant quantity " refers to the amount that the patient is produced desired interests.Amount will change between individuality and individuality and will decide on a large amount of factors of the potential cause of disease of the total physical appearance that comprises the patient and symptom to be treated.The volume production that is used for the hGH polypeptide of therapy is given birth to acceptable change rate and desired reaction is maintained under the useful level.The treatment significant quantity of the present composition can easily be passed through the those skilled in the art, uses open available material and program to determine.
Water-soluble polymers can be to include, but is not limited to straight chain, bifurcated or ramose any structure form.Usually, water-soluble polymers is such as poly-(ethylene glycol) poly-(alkylene glycol) (PEG), but also can use other water-soluble polymerss.For example, PEG is in order to describe some embodiment of the present invention.
PEG be know, water-soluble polymers, it is commercially available or can be according to the method known to the those skilled in the art, by ring-opening polymerization preparation (Sandier and the Karo of ethylene glycol, Polymer Synthesis, Academic Press, New York, the 3rd volume, the 138-161 page or leaf).Term " PEG " is extensively in order to contain any peg molecule, do not consider size or the terminal modification of PEG, and can be expressed as with the hGH polypeptide by following formula and be connected: XO-(CH 2CH 2O) n-CH 2CH 2-Y, wherein n be 2 to 10,000 and X be H or include, but is not limited to C 1-4Alkyl, protecting group or end modified group end modified.
In some cases, be used for PEG of the present invention and stop by hydroxyl or methoxyl group, that is to say that X is H or CH at an end 3(" methoxyl group PEG ").Perhaps, PEG can be stopped by reactive group, and then forms the double functional copolymer.Typical reactive group can comprise commonly used (to include, but is not limited to the described reactive group that is found in 20 kinds of functional group reactionses in the common amino acid, maleimide base group, activated carbonic ether (including, but is not limited to the p-nitrophenyl ester), activated ester (include, but is not limited to, N-maloyl imines, p-nitrophenyl ester) and aldehyde), and to 20 kinds of common amino acids be inertia but with the functional group that is present in the complementary functional groups specific reaction in the non-naturally encoded amino acid.Should be appreciated that another of the PEG that is showed by Y is terminal will directly or indirectly to be connected in the hGH polypeptide by naturally occurring or non-naturally encoded amino acid in following formula.In certain embodiments, strong nucleophile (includes, but is not limited to, hydrazine, hydrazides, azanol, Urea,amino-) can be in due course be present in aldehydes or ketones radical reaction in the non-naturally encoded amino acid to form hydrazone, oxime or semicarbazone, in some cases, it can be further by being reduced by the processing of suitable reductive agent.Perhaps, strong nucleophile can be incorporated in the hGH polypeptide by non-naturally encoded amino acid, and in order to preferentially to react with the ketone or the aldehyde radical that are present in the water-soluble polymers.
Any molecular weight of PEG can use by actual requirement, and it includes, but is not limited to about 100 dalton (Da) to 100,000Da or be more (including, but is not limited to, is 0.1-50kDa or 10-40kDa sometimes) on request.The molecular weight of PEG can have and includes, but is not limited at about 100Da and about 100 000Da or 100, the wide region between 000Da is above.The molecular weight of PEG can between the 000Da, include, but is not limited to 100 at about 100Da and about 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of PEG is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of PEG is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of PEG is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of PEG is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of PEG is about 10, and 000Da and 40 is between the 000Da.Also can use side chain PEG, it includes, but is not limited to, and has the PEG molecule that has at each chain of the MW of 1-100kDa (including, but is not limited to 1-50kDa or 5-20kDa) range.The molecular weight of side chain PEG can (include, but is not limited to) about 1,000Da and about 100, and 000Da or 100 is between 000Da is above.The molecular weight of side chain PEG can be about 1, and 000Da and about 100 between the 000Da, includes, but is not limited to, and 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da and 1,000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 20 is between the 000Da.The PEG molecule of broad range is described in and includes, but is not limited to the Shearwater Polymers, Inc.catalog, and among the NektarTherapeutics catalog, it incorporates this paper by reference into.
Usually, the PEG molecule at least one end can be used for and the reaction of non-naturally encoded amino acid.In certain embodiments, the hGH polypeptide variants with PEG derivative contain can with the chemical functional group who is present in the chemical functional group's reaction on the non-naturally encoded amino acid whose side chain.
The main polymer chain of water-soluble polymers can be poly-(ethylene glycol).Yet, should be appreciated that to include, but is not limited to gather (ethylene glycol) and comprise poly-(dextran) and the multiple water-soluble polymers of other related polymers of poly-(propylene glycol) is applicable to that also the use of practice of the present invention and term PEG or poly-(ethylene glycol) is to be used for containing and to comprise all described molecules.Term PEG includes, but is not limited to be its any type of poly-(ethylene glycol), PEG, the bifurcated PEG, the PEG of branch, the side joint PEG that comprise difunctionality PEG, multi-arm PEG, derivatize (that is to say, PEG or have the related polymer of one or more side joints in the functional group of main polymer chain), or wherein have the PEG of degradable key.
PEG normally transparent, colourless, tasteless, water-soluble, thermally-stabilised to adding, to many chemical reagent inert, not hydrolysis or degeneration, and normally nontoxic.Poly-(ethylene glycol) is considered to can be biocompatible, that is to say, PEG can coexist and can not work the mischief with living tissue or organism.More particularly, PEG is a non-immunogenic substantially, that is to say that PEG does not trend towards producing in vivo immune response.When the branch period of the day from 11 p.m. to 1 a.m with some required functions that is connected in vivo such as biologically active agent, PEG trends towards covering medicament and can reduce or eliminate any immune response so that the existence of organism tolerable medicament.The PEG binding substances can not trend towards producing the immune response of essence or causing blood coagulation or other ill effects.Has formula-CH 2CH 2O-(CH 2CH 2O) n--CH 2CH 2-PEG be applicable to the present invention, wherein n is about 3 to about 4000, is generally about 20 to about 2000.In some embodiments of the invention, have about 800Da to about 100, the PEG of the molecular weight of 000Da is particularly suited for as main polymer chain.The molecular weight of PEG can have and includes, but is not limited at about 100Da and about 100 000Da or 100, the wide region between 000Da is above.The molecular weight of PEG can between the 000Da, include, but is not limited to 100 at about 100Da and about 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of PEG is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of PEG is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of PEG is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of PEG is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of PEG is about 10, and 000Da and 40 is between the 000Da.
That main polymer chain can be straight chain or ramose.The branched polymer main chain is known usually in affiliated field.Usually, branched polymer has center branch core and a plurality of straight-chain polymer chains that are connected with the center branch core.PEG normally uses with branch's form, and it can prepare by the various polyvalent alcohols that oxyethane added to such as glycerine, glycerine oligomer, tetramethylolmethane and Sorbitol Powder.The center branch part also can be obtained by the some amino acid such as Methionin.Ramose poly-(ethylene glycol) can be by general formula R (PEG-OH) mExpression, wherein R is derived from the core such as glycerine, glycerine oligomer or tetramethylolmethane, and m represents the quantity of arm.Such as United States Patent (USP) the 5th, 932,4625,643,575; 5,229,490; 4,289, No. 872; U.S. patent application case 2003/0143596; WO 96/21469; Also can be used as main polymer chain with the multi-arm PEG molecule of the described PEG molecule of describing among the WO 93/21259, described each patent is all to incorporate this paper by reference into.
The PEG of branch also can be by PEG (--YCHZ 2) nThe bifurcated PEG form of expression, wherein Y is that linking group and Z are the activated terminal group that is connected with CH by a string atom with specified length.
Another kind of branch form, i.e. side joint PEG has along the PEG main chain rather than at the reactive group such as carboxyl of the end of PEG chain.
Except that the form of described PEG, polymkeric substance also can have weak or degradable key through preparation in main chain.For example, the PEG ester bond that can in main polymer chain, have the hydrolytic action of being subject to through preparation.As follows, described hydrolytic action can cause polymer cracking to become to have the fragment of lower molecular weight:
-PEG-CO 2-PEG-+H 2O→PEG-CO 2H+HO-PEG-
It will be understood by one of ordinary skill in the art that term poly-(ethylene glycol) or PEG represent or comprise the known form of ownership that includes, but is not limited to described form disclosed herein in the affiliated field.
Many other polymkeric substance also are applicable to the present invention.In certain embodiments, have 2 water miscible main polymer chains and be particularly useful for the present invention to about 300 ends.The example of the polymkeric substance that is fit to includes, but is not limited to other poly-(alkylene glycols) such as poly-(propylene glycol) (" PPG "), and its multipolymer (including, but is not limited to the multipolymer of ethylene glycol and propylene glycol), its trimer, its mixture are like that.Although the molecular weight of each chain of main polymer chain can change, its normally at about 800Da to about 100, in the scope of 000Da, usually about 6,000Da is to about 80, in the scope of 000Da.The molecular weight of each chain of main polymer chain can between the 000Da, include, but is not limited at about 100Da and about 100,100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of each chain of main polymer chain is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of each chain of main polymer chain is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of each chain of main polymer chain is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of each chain of main polymer chain is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of each chain of main polymer chain is about 10, and 000Da and 40 is between the 000Da.
Those skilled in the art will realize that the aforementioned inventory of water-soluble main chain is not to be illustrative completely and only substantially, and have all polymeric materials of above-mentioned character and contained because being applicable to the present invention.
In some embodiments of the invention, polymer derivant is " polyfunctional ", the meaning be main polymer chain have at least two through functional group functionalized or activatory end, and may as many as about 300 ends.Multifunctional polymer derivant includes, but is not limited to have the straight-chain polymer of two ends, and each end is bonded to and can be identical or different functional groups.
Water-soluble polymers can be connected with hGH polypeptide of the present invention.Water-soluble polymers can be by incorporating the non-naturally encoded amino acid in the hGH polypeptide into, or any functional group or substituting group non-naturally encoded or natural amino acids coding, or add any functional group or substituting group non-naturally encoded or natural amino acids coding to and connect.Perhaps, water-soluble polymers be by naturally occurring amino acid (including, but is not limited to the amino of halfcystine, Methionin or N-terminal residue) with incorporate non-naturally encoded amino acid whose hGH polypeptide into and be connected.In some cases, hGH polypeptide of the present invention comprises 1,2,3,4,5,6,7,8,9,10 kind of non-natural amino acid, one of them or more than one non-naturally encoded amino acid be connected in water-soluble polymers (including, but is not limited to PEG and/or oligosaccharides).In some cases, hGH polypeptide of the present invention comprise 1,2,3,4,5,6,7,8,9 in addition, the natural amino acids coding that is connected with water-soluble polymers more than 10 kind or 10 kinds.In some cases, hGH polypeptide of the present invention comprises one or more non-naturally encoded amino acid that is connected with water-soluble polymers, with one or more naturally occurring amino acid that is connected with water-soluble polymers.In certain embodiments, with respect to non-binding form, be used for the serum half-life that water-soluble polymers of the present invention strengthens the hGH polypeptide.
The quantity of the water-soluble polymers that is connected with hGH polypeptide of the present invention (that is to say Pegylation or glycosylated degree) can be through regulating with (include, but is not limited to increase or reduce) that change is provided such as the characteristic of pharmacological, pharmacokinetics or the drug effect of transformation period in vivo.In certain embodiments, the more non-modified polypeptide increase of the transformation period of hGH is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2 times, 5 times, 10 times, 50 times or at least about 100 times.
Degree that water-soluble polymers is connected with the hGH polypeptide and position can be regulated the hGH polypeptide receptor at hGH polypeptide and 1 place, position or combine the combination of collocation thing.In certain embodiments, key through arranging in case the hGH polypeptide with about 400nM or the K below the 400nM d, with 150nM or the K below the 150nM dAnd in some cases, with 100nM or the K below the 100nM dThe hGH polypeptide receptor at combining site 1 place, K dAccording to such as people such as Spencer, J.Biol.Chem., the balance described in the 263:7862-7867 (1988) is measured in conjunction with calibrating.
Be used for activated polymer and be used for the method for binding peptide and chemical action is described in document and in affiliated field for known.The common method that is used for activated polymer includes, but is not limited to, and waits with cyanogen bromide, periodate, glutaraldehyde, di-epoxide, Epicholorohydrin, divinyl sulfone, carbodiimide, alkylsulfonyl halogenide, three chlorotriazines to activate functional group.(referring to, R.F.Taylor, (1991), PROTEIN IMMOBILISATION.FUNDAMENTALAND APPLICATIONS, Marcel Dekker, N.Y.; S.S.Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; People such as G.T.Hermanson, (1993), IMMOBILIZED AFFINITY LlGAND TECHNIQUES, AcademicPress, N.Y.; Dunn, people such as R.L. compile .POLYMERIC DRUGS AND DRUG DELIVERYSYSTEMS, ACS Symposium Series the 469th volume, American Chemical Society, Washington, D.C.1991).
The comment and the disquisition of some functionalized and keying actions to PEG are available.Referring to (for example), Harris, Macromol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); People such as Wong, Enzyme Microb.Technol.14:866-874 (1992); People such as Delgado, CriticalReviews in Tlierapeutic Drug Carrier Systems 9:249-304 (1992); Zalipsky, BioconjugateChem.6:150-165 (1995).
The method that is used for activated polymer also is found in WO 94/17039, United States Patent (USP) the 5th, 324, No. 844, WO94/18247, WO 94/04193, United States Patent (USP) the 5th, 219, No. 564, United States Patent (USP) the 5th, 122, No. 614, WO90/13540, United States Patent (USP) the 5th, 281, No. 698 and WO 93/15189, and can find the method for the keying action between the enzyme that is used for activated polymkeric substance and includes, but is not limited to following enzyme: blood coagulation factor VIII (WO 94/15625), oxyphorase (WO 94/09027), the molecule the (the 4th that has oxygen, 412, No. 989 United States Patent (USP)s), rnase and hemocuprein (people such as Veronese, App.Biochem.Biotech.11:141-52 (1985)).The reference of all references and patent are to incorporate this paper by reference into.
Reaction product stands the hydrophobic interaction chromatogram subsequently so that separate with free PEG through the hGH of Pegylation polypeptide variants.The condition that is fit to is looked the relative dimension of the desired relatively binding substances of cross-linked composite and is changed, and easily determines by the those skilled in the art.The eluant that contains desired binding substances can be by ultrafiltration and concentration and by the diafiltration desalination.
In case of necessity, from the hydrophobicity chromatogram obtain through the hGH of Pegylation polypeptide, can be in addition by including, but is not limited to one or more program purifying known to the those skilled in the art that are of following program: affinity chromatography; Negatively charged ion or cation-exchange chromatography (use includes, but is not limited to DEAE SEPHAROSE); The silica chromatography; Anti-phase HPLC; Gel-filtration (use includes, but is not limited to SEPHADEX G-75); The hydrophobic interaction chromatography; Size-exclusion chromatography; Metal chelate chromatography; Ultrafiltration/diafiltration; Ethanol sedimentation; Ammonium sulfate precipitation; Chromatofocusing; Displacement chromatography; Electrophoretic procedures (including, but is not limited to the isoelectrofocusing of preparation property), difference solubility (including, but is not limited to ammonium sulfate precipitation) or extraction method.Apparent molecular weight can be estimated (Preneta, AZ in PROTEIN PURIFICATION METHODS, A PRACTICALAPPROACH (Harris ﹠amp by GPC is compared with the globular proteins standard; Angal compiles) IRL Press 1989,293-306).The purity of hGH-PEG binding substances can be by separating proteolytic degradation (including, but is not limited to the trypsinase cracking), and then mass spectroscopy is assessed.PepinskyRB. wait people, J.Pharmcol.﹠amp; Exp.Ther.297 (3): 1059-66 (2001).
The water-soluble polymers that is connected with the amino acid of hGH polypeptide of the present invention can be unrestrictedly in addition through deriving or replacing.
Other PEG derivatives and general Pegylation technology
The exemplary PEG molecule of other that can be connected with the hGH polypeptide, and the Pegylation method comprises described PEG molecule and the Pegylation method that is described in (for example) following patent: U.S. Patent Publication case the 2004/0001838th; 2002/0052009; 2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447; 2003/0158333; 2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047; 2002/0099133; 2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430; 2002/0040076; 2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526; No. 2001/0021763; United States Patent (USP) the 6th, 646,110; 5,824,778; 5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502; 5,281,698; 5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167; 6,610,281; 6,515,100; 6,461,603; 6,436,386; 6,214,966; 5,990,237; 5,900,461; 5,739,208; 5,672,662; 5,446,090; 5,808,096; 5,612,460; 5,324,844; 5,252,714; 6,420,339; 6,201,072; 6,451,346; 6,306,821; 5,559,213; 5,747,646; 5,834,594; 5,849,860; 5,980,948; 6,004,573; 6,129, No. 912; WO97/32607, EP 229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921131, WO 98/05363, EP 809996, WO 96/41813, WO 96/07670, EP 605963, EP 510356, EP 400472, EP 183503 and EP 154316, described patent is to incorporate this paper by reference into.Any form strand, side chain, multi-arm chain, simple function, dual functional, multifunctional form or its any combination that can include, but is not limited to of any PEG molecule of Miao Shuing is used herein.
Enhancing is to sero-abluminous avidity
Also various molecules can be fused to hGH polypeptide of the present invention to regulate the hGH transformation period of polypeptide in serum.In certain embodiments, molecule is connected in or is fused to hGH polypeptide of the present invention to strengthen the sero-abluminous avidity of the endogenous of animal.
For example, in some cases, produce the reorganization syzygy of hGH polypeptide and albumin bound sequence.Exemplary albumin bound sequence includes, but is not limited to albumin bound territory from suis property protein G (referring to (for example), people such as Makrides, people such as J.Pharmacol.Exp.Ther.277:534-542 (1996) and Sjolander, J, Immunol.Methods 201:115-123 (1997)), or albumin-binding peptide, such as being described in people such as (for example) Dennis, the described albumin-binding peptide among the J.Biol.Chem.277:35035-35043 (2002).
In other embodiments, hGH polypeptide of the present invention is through the lipid acid acidylate.In some cases, lipid acid promotes to combine with sero-abluminous.Referring to (for example), people such as Kurtzhals, Biochem.J.312:725-731 (1995).
In other embodiments, hGH polypeptide of the present invention is direct and serum albumin (including, but is not limited to human serum albumin) fusion.Those skilled in the art will realize that multiple other molecules also can be connected with hGH of the present invention to regulate the combination to serum albumin or other serum compositions.
The glycosylation of XVI.hGH polypeptide
The present invention includes and incorporate the non-naturally encoded amino acid whose hGH polypeptide that one or more have the carbohydrate residue into.The carbohydrate residue can be natural (including, but is not limited to the N-acetylglucosamine) or non-natural (including, but is not limited to 3-fluorine semi-lactosi).Carbohydrate glycosidic linkage (including, but is not limited to N-ethanoyl semi-lactosi-L-Serine) that can connect by N-or that O-connects or non-natural key (including, but is not limited to oxime or corresponding C-glucosides that connect or that S-connects) are connected with non-naturally encoded amino acid.
Carbohydrate (including, but is not limited to glycosyl) part can be in vivo or is added the hGH polypeptide in vitro to.In some embodiments of the invention, comprise the non-naturally encoded amino acid whose hGH polypeptide that contains carbonyl be through modify with amino oxygen base group deutero-carbohydrate with produce the correspondence that connects by the oxime key through glycosylated polypeptide.In case be connected with non-naturally encoded amino acid, carbohydrate just can be in addition by making with extra care the oligosaccharides that is incorporated into the hGH polypeptide with generation with the processing of glycosyltransferase and other enzymes.Referring to (for example), people J.Am.Chem.Soc.125:1702-1703 (2003) such as H.Liu.
XVII.GH supergene family member's dimer and polymer
The present invention also provides GH supergene family member combination (including, but is not limited to hGH and hGH analogue), (that is to say such as homodimer, heterodimer, homology polymer or heteromultimers, tripolymer, the tetramer etc.), wherein be incorporated into another GH supergene family member or its variant or be any other polypeptide of non-GH supergene family member or its variant such as the GH supergene family member polypeptide that contains one or more non-naturally encoded amino acid whose hGH, it is directly to be incorporated into the polypeptide main chain or by the connexon combination.Because it compares the molecular weight of increase with monomer, so the GH supergene family member such as hGH, dimer or polymer binding substances can show novel or required character, include, but is not limited to different pharmacology, pharmacokinetics, drug effect, treatment transformation period or plasma half-life through regulating through regulating with respect to monomer GH supergene family member.In certain embodiments, will regulate the dimerization of GH supergene family member acceptor such as hGH of the present invention, dimeric GH supergene family member.In other embodiments, GH supergene family member's dimer of the present invention or polymer will serve as GH supergene family member receptor antagonist, agonist or modulator.
In certain embodiments, one or more are present in the hGH molecule that contains among dimer or the polymeric hGH and comprise the non-naturally encoded amino acid that the water-soluble polymers interior with being present in position II calmodulin binding domain CaM is connected.Thereby each molecule of dimer or polymeric hGH molecule is easy to be incorporated into the hGH polypeptide receptor by I interface, position, but can not be incorporated into the 2nd hGH polypeptide receptor by II interface, position.Therefore, hGH polypeptide dimer or polymer can engage the position I combining site of two kinds of each acceptors in the different hGH polypeptide receptor, but,, the hGH polypeptide receptor serves as the hGH polypeptide antagonist so can not engaging the II zone, position of hGH polypeptide ligand and dimer or polymer because the hGH molecule has and is connected in the amino acid whose water-soluble polymers that is present in the non-genetic coding in the II zone, position.In certain embodiments, one or more are present in the hGH molecule that contains in dimer or the polymeric hGH polypeptide and comprise the non-naturally encoded amino acid that the water-soluble polymers interior with being present in position I calmodulin binding domain CaM is connected, and make it possible to be incorporated into II zone, position.Perhaps, in certain embodiments, one or more are present in the hGH molecule that contains in dimer or the polymeric hGH polypeptide and comprise and be present in the non-naturally encoded amino acid that the water-soluble polymers that is not the position in position I or position II calmodulin binding domain CaM is connected, so that the both can be used for combination.In certain embodiments, use the combination of hGH molecule so that position I, position II or both can be used for combination.At least a have can be used for bonded position I and at least a combination with the hGH molecule that can be used for bonded position II can provide the molecule with desired activity or character.In addition, the combination with the hGH molecule that can be used for bonded position I and position II can produce super agonist hGH molecule.
In certain embodiments, GH supergene family member polypeptide is that directly (including, but is not limited to) connects by Asn-Lys amido linkage or Cys-Cys disulfide linkage.In certain embodiments, GH supergene family member's polypeptide through connecting and/or the non-GH supergene family member through connecting will comprise different non-naturally encoded amino acid to promote dimerization.
Perhaps, two kinds of GH supergene family member's polypeptide and/or the non-GH supergene family member through connecting connect by connexon.Any allos or homology difunctionality connexon can be in order to connecting two kinds of GH supergene family members and/or the non-GH supergene family member through connecting, polypeptide, and it can have identical or different elementary sequences.In some cases, in order to GH supergene family member and/or the non-GH supergene family member through connecting, the connexon that polypeptide is strapped in together can be difunctionality PEG reagent.Connexon can have the molecular weight or the molecular length of broad range.Big or can be in order to provide desired spatial relationship or conformation between hGH and the entity through being connected than the connexon of small molecular weight.
In certain embodiments, the invention provides the water-soluble difunctionality connexon with dumbbell structure, it comprises: a) first functional group on first end of main polymer chain at least; And b) at least one second functional group on second end of main polymer chain.Second functional group can be identical or different with first functional group.In certain embodiments, second functional group can not with first functional group reactions.In certain embodiments, the invention provides the water-soluble cpds of at least one arm that comprises the branching molecule structure.For example, the branching molecule structure can be dendritic.In certain embodiments, the invention provides by reacting the polymer that forms with water-soluble activated polymer, it comprises one or more GH supergene family members such as hGH.
XVIII. offer medicine and medical composition
Polypeptide of the present invention or protein (include, but is not limited to hGH, synthetic enzyme, comprise the protein of one or more alpha-non-natural amino acids etc.) optionally (include, but is not limited to) make up to be used for the treatment of purposes with the medical supporting agent that is fit to.For example, described composition comprises the compound for the treatment of significant quantity and pharmaceutically acceptable supporting agent or vehicle.Described supporting agent or vehicle include, but is not limited to salt solution, buffer saline, glucose, water, glycerine, ethanol and/or its combination.Make prescription be suitable for the dispensing pattern.Generally speaking, throw with method of protein be known to the those skilled in the art and applicable to the dispensing of polypeptide of the present invention.
(optionally) according to being method known to the those skilled in the art, in one or more suitable in vitro and/or in vivo animal disease models, test comprises the therapeutic composition of one or more polypeptide of the present invention to confirm effect, tissue metabolism and estimation dosage.Specifically, dosage can be at first alpha-non-natural amino acid by herein (include, but is not limited to respect to the natural amino acid homologue, modified with the hGH polypeptide that comprises one or more alpha-non-natural amino acids and the comparison of natural amino acid hGH polypeptide) activity, stability or other be fit to measure, that is to say in relevant calibrating and determined.
Dispensing is the molecule introducing to be offerd medicine with blood or the histiocytic final any route that contacts by being usually used in.Non-natural amino acid polypeptides of the present invention is optionally with one or more pharmaceutically acceptable supporting agents, throw in any suitable manner with.The method that is fit to of the described polypeptide in patient's throwing and context of the present invention is an available, and, although can use more than one route to throw and concrete composition, concrete route can often provide than more instant and more effective effect of another kind of route or reaction.
Pharmaceutically acceptable supporting agent be partly throws by institute and concrete composition and by determining in order to the throwing and the concrete method of composition.Therefore, the multiple suitable prescription that has medical composition of the present invention.
HGH polypeptide of the present invention, can by any habitual route that includes, but is not limited to parenteral route that is suitable for protein or peptide throw with, for example by include, but is not limited to hypodermically or the injection of intravenous injection or any other form or transfusion throw with.Peptide composition can by include, but is not limited to per os, intravenously, peritonaeum Inner, intramuscular, through skin, subcutaneous, local, hypogloeeis or rectal multiple route throw with.The composition that comprises modified or not modified non-natural amino acid polypeptide also can by liposome throw with.Described dispensing route and proper formulation are generally known to the those skilled in the art.Comprise that non-naturally encoded amino acid whose hGH polypeptide can use separately or use with other combination of components that are fit to such as medical supporting agent.
Also can with independent or with other combination of components that are fit to comprise non-natural amino acid whose hGH polypeptide make treat by suck throw and aerosol formulations (that is to say that it can be " atomizing ").Aerosol formulations can be placed on such as Refrigerant 12, propane, nitrogen, suchlike in the acceptable propelling agent of pressurization.
Be suitable for (such as) by the non-prescription through the intestines dispensing of intraarticular (in the joint), intravenously, intramuscular, skin Inner, peritonaeum Inner and subcutaneous route mode, comprise can contain antioxidant, buffer reagent, fungistat and make the solute that prescription and expection recipient's blood etc. opens water-based and non-aqueous, etc. open aseptic injectable solution and can comprise the water-based and the non-aqueous sterile suspensions of suspension agent, solubilizing agent, thickening material, stablizer and sanitas.The prescription of hGH can be present in such as in the unitary dose of ampoule and bottle or the multiple doses sealed vessel.
Non-is preferred medication administration method through intestines dispensing and intravenously dispensing.Specifically, the dispensing route that has been used for natural amino acid homologue therapeutical agent (includes, but is not limited to, be generally used for EPO, GH, G-CSF, GM-CSF, IFN, interleukin, antibody and/or any other proteinic described dispensing route that pharmaceutically transmits), be provided for the preferred dispensing route and the prescription of polypeptide of the present invention together with the prescription of current use.
In the context of the present invention, throw with patient's dosage and decide, be enough in the patient, have useful therapeutic response in time on using, or other suitable activity.Dosage is the symptom by the activity of the effect of concrete carrier or prescription and employed non-natural amino acid polypeptides, stability or serum half-life and patient, and patient's to be treated body weight or body surface area are determined.The size of dosage is also determined by existence, the nature and extent of any opposite side effect of the dispensing of following concrete carrier, prescription or the like in concrete patient.
Treating in the treatment of determining disease (including, but is not limited to cancer, heredopathia, diabetes, AIDS or the like) or prevention throw and carrier or during the significant quantity of prescription, the doctor evaluates the process of circulating plasma level, prescription toxicity, disease and/or the production of (when relevant) anti-non-natural amino acid polypeptides antibody.
(for example) in the scope that is equivalent to the proteinic dosage of present employed treatment, it is through adjusting to change compositions related activity or serum half-life usually for throwing and 70 kilograms of patients' dosage.Carrier of the present invention or medical prescription can be by any known habitual therapies, comprise the dispensing of antibody dispensing, vaccine dispensing, cytotoxic agent, natural amino acid polypeptide, nucleic acid, nucleotide analog, biological response modifier, like thatly come the supplement therapy condition.
Be dispensing, prescription of the present invention is with by the LD-50 of relevant prescription or ED-50 and/or under various concentration, (including, but is not limited to) when quality that is applicable to the patient and general health, the determined rate of application of the observation of any side effect of non-natural amino acid polypeptides throw with.Dispensing can be finished by single dose or separate doses.
If experience is injected the patient infection of prescription and had a fever, catches cold or myalgia, he can accept Asprin (aspirin), Ibuprofen BP/EP (ibuprofen), ethanamide phenol or other pain/fever control medicine of suitable dosage so.Experience such as fever, myalgia and the patient of reaction to transfusion who catches cold before the transfusion in future, premedicated 30 minutes with Asprin, ethanamide phenol or (including, but is not limited to) diphenhydramine (diphenhydramine).Dolantin (Meperidine) is to be used for more serious the catching cold and myalgia that can not react fast to febrifuge and antihistamine.The seriousness of visual response and slow down or interrupt cell and inject.
Human hGH polypeptide of the present invention can directly be thrown and the Mammals person under inspection.Dispensing is to be undertaken by any route that is generally used for the hGH polypeptide is introduced the person under inspection.Although under any given situation, optimal route will be decided on the character and the seriousness of the symptom of being treated, but the hGH peptide composition comprises the described composition that is suitable for following dispensing according to an embodiment of the invention: per os, rectum, local, suck (including, but is not limited to pass through aerosol), cheek (including, but is not limited to the hypogloeeis), vagina, non-ly (include, but is not limited to subcutaneous through intestines, intramuscular, skin Inner, intraarticular, in the pleura, peritonaeum Inner, in the brain, intra-arterial or intravenously), local (that is to say, skin and mucomembranous surface comprise airway surface) and offer medicine through skin.Dispensing can be partial or whole body.The prescription of compound can be present in such as in the unitary dose of ampoule and bottle or the multiple doses sealed vessel.HGH polypeptide of the present invention can be prepared in and be in the unitary dose injectable forms mixture with pharmaceutically acceptable supporting agent (including, but is not limited to solution, suspension or emulsion).HGH polypeptide of the present invention also can by inject continuously (minipump that uses (including, but is not limited to) such as osmotic pump), single bolus or slowly discharge store prescription throw with.
The prescription that is suitable for offeing medicine comprise can contain antioxidant, buffer reagent, fungistat and make the water-based of the solute that prescription etc. opens and non-aqueous solution, etc. open sterile solution and can comprise the water-based and the non-aqueous sterile suspensions of suspension agent, solubilizing agent, thickening material, stablizer and sanitas.Sterilized powder, particle and the tablet preparation of the kind that solution and suspension can be described from preamble.
Lyophilize is to be used to present proteinic common technology, and it is used for removing from the protein formulation paid close attention to and anhydrates.To be material to be dried be frozen earlier then by the distillation in vacuum environment to remove the method for deicing or chilled solvent by it for lyophilize or lyophilization.Vehicle can be included in the freeze dried in advance prescription, to strengthen in the stability during the freezing dry process and/or the stability after improving the freeze-drying prods storage.Pikal, people Pharm.Res.8 (3): 285-291 (1991) such as M.Biopharm.3 (9) 26-30 (1990) and Arakawa.
The spraying drying of pharmaceuticals also is known to the those skilled in the art.For example, referring to Broadhead, people such as J., " The Spray Drying of Pharmaceuticals, " in Drug Dev.Ind.Pharm, 18 (11 ﹠amp; 12), 1169-1206 (1992).Except that the small molecules pharmaceuticals, various biomaterial spraying dryings and described biomaterial are comprised: enzyme, serum, blood plasma, microorganism and yeast.Spraying drying is an otherwise effective technique, because it can be in the single stage method changes into liquid pharmaceutical formulation meticulous, dustless or agglomerating powder.Basic fundamental comprises following four steps: a) feedstock solution is atomized into spraying; B) spraying-air contact; C) dry spraying; With d) desciccate is separated with dry air.The United States Patent (USP) the 6th, 235,710 and 6,001 of incorporating this paper by reference into, No. 800 descriptions prepare recombinant erythropoietin by spraying drying.
Medical composition of the present invention and prescription can comprise pharmaceutically acceptable supporting agent, vehicle or stablizer.Pharmaceutically acceptable supporting agent be partly throws by institute and concrete composition and by determining in order to the throwing and the concrete method of composition.Therefore, there is the medical composition of the present invention multiple suitable prescription (referring to (for example) Remington ' s Pharmaceutical Sciences, the 17th edition, 1985) of (comprising optionally pharmaceutically acceptable supporting agent, vehicle or stablizer)).
The supporting agent that is fit to includes, but is not limited to, and contains succinate, phosphoric acid salt, borate, HEPES, Citrate trianion, Histidine or histidine derivative, imidazoles, acetate, supercarbonate and other organic acid damping fluids; Include, but is not limited to the antioxidant of xitix; Low molecular weight polypeptide includes, but is not limited to the described polypeptide less than about 10 residues; Protein includes, but is not limited to serum albumin, gelatin or immunoglobulin (Ig); Include, but is not limited to the hydrophilic polymer of polyvinylpyrrolidone; Amino acid includes, but is not limited to glycine, glutamine, Histidine or histidine derivative, methionine(Met), asparagine, arginine, L-glutamic acid or Methionin; Monose, disaccharides and other carbohydrates include, but is not limited to trehalose, sucrose, glucose, seminose or dextrin; Include, but is not limited to the sequestrant of EDTA; Divalent-metal ion includes, but is not limited to zinc, cobalt or copper; Include, but is not limited to the sugar alcohol of mannitol or Sorbitol Powder; Include, but is not limited to the salify gegenion of sodium; And/or nonionic surface active agent, include, but is not limited to Tween TM(include, but is not limited to Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20; PS20)), Pluronics TMWith other Pluronic acid (pluronic acid), include, but is not limited to Pluronic acid F68 (poloxamer (poloxamer) 188) or PEG.The tensio-active agent (for example) that is fit to includes, but is not limited to based on poly-(oxyethane)-poly-(propylene oxide)-poly-(oxyethane), that is to say, (PEO-PPO-PEO) polyethers, or based on poly-(propylene oxide)-poly-(oxyethane)-poly-(bad Ethylene Oxide), that is to say, (PPO-PEO-PPO) polyethers, or its combination.PEO-PPO-PEO and PPO-PEO-PPO are with trade name Pluronics TM, R-Pluronics TM, Tetronics TMAnd R-Tetronics TM(BASF Wyandotte Corp., Wyandotte, Mich) commercially available, and be described in addition in No. the 4th, 820,352, the United States Patent (USP) all incorporating this paper by reference into.Other ethylene/polypropylene block polymers can be the tensio-active agents that is fit to.The combination of tensio-active agent or tensio-active agent can be used for stable hGH through Pegylation, and opposing includes, but is not limited to by one or more stress that stir the stress that produces.Some above-mentioned tensio-active agents can be called " weighting agent ".Some also can be called " tension force modification agent ".
The hGH polypeptide of the present invention that comprises the described polypeptide that is connected with water-soluble polymers such as PEG also can by sustained release system or as the part of sustained release system throw with.Sustained-release composition comprises that (including, but is not limited to) is the semi permeable polymeric matrix of the formed article form that includes, but is not limited to film or microcapsule.Lasting release matrix comprises biocompatible material, such as poly-(methacrylic acid 2-hydroxyl ethyl ester) (people such as Langer, J.Biomed.Mater.Res., 15:267-277 (1981); Langer, Chem.Tech., 12:98-105 (1982)), ethylene vinyl acetate people such as (, the same) Langer or poly--D-(-)-3-hydroxybutyric acid (EP 133,988), polylactide (poly(lactic acid)) (United States Patent (USP) the 3rd, 773, No. 919; EP 58,481), the multipolymer of poly-glycollide (polymkeric substance of oxyacetic acid), polylactide copolymerization glycollide (multipolymer of lactic acid and oxyacetic acid) polyanhydride, L-L-glutamic acid and γ-ethyl-L-L-glutamic acid (people such as Sidman, Biopolymers, 22,547-556 (1983)), poly-(ortho acid) ester, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, lipid acid, phosphatide, polysaccharide, nucleic acid, polyamino acid, amino acid, such as phenylalanine, tyrosine, L-iLeu, polynucleotide, polyethylene propylene, polyvinylpyrrolidone and silicone.Sustained-release composition also comprises liposome trapping compound.The compound that contains liposome is by known method preparation itself: DE 3,218, and 121; People such as Eppstein, Proc.Natl.Acad.Sci.U.S.A., 82:3688-3692 (1985); People such as Hwang, Proc.Natl.Acad.Sci.U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; United States Patent (USP) the 4th, 619, No. 794; EP143,949; United States Patent (USP) the 5th, 021, No. 234; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485,045 and 4,544, No. 545; With EP 102,324.The reference of all references and patent are to incorporate this paper by reference into.
HGH polypeptide through the liposome trapping can be by being described in (for example) DE 3,218,121; People such as Eppstein, Proc.Natl.Acad.Sci.U.S.A., 82:3688-3692 (1985); People such as Hwang, Proc.Natl.Acad.Sci.U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; United States Patent (USP) the 4th, 619, No. 794; EP 143,949; United States Patent (USP) the 5th, 021, No. 234; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485,045 and 4,544, No. 545; With the method preparation among the EP 102,324.The composition of liposome and size be know or can be easily rule of thumb determine by the those skilled in the art.Some examples of liposome are as people such as (for example) Park JW, Proc.Natl.Acad.Sci.USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D (volume): MEDICALAPPLICATIONS OF LIPOSOMES (1998); People such as Drummond DC, Liposomal drug deliverysystems for cancer therapy, in Teicher B (volume): CANCER DRUG DISCOVERY ANDDEVELOPMENT (2002); People such as Park JW, Clin.Cancer Res.8:1172-1181 (2002); People such as Nielsen UB, Biochim.Biophys.Acta 1591 (1-3): 109-118 (2002); People such as Mamot C are described in the Cancer Res.63:3154-3161 (2003).The reference of all references and patent are to incorporate this paper by reference into.
In the context of the present invention, throwing and patient's dosage should be enough to cause useful reaction in time in the person under inspection.Usually, although described significant quantity is easy to be treated the control of judgement, but each dosage non-through intestines throw and total medicine and pharmacology significant quantity of hGH polypeptide of the present invention be every day the per kilogram weight in patients about 0.01 μ g to about 100 μ g, or the about 0.05mg of per kilogram weight in patients arrives in the scope of about 1mg.The frequency of administration also is subject to treat the control of judgement, and can have the faster or slower frequency of commercially available hGH polypeptide products frequency that is used for the mankind than approval.Usually, of the present invention through the hGH of Pegylation polypeptide can by above-mentioned any dispensing route throw with.
It is believed that, use previous description those skilled in the art can at utmost utilize the present invention.Following example only is illustrative, in any case and limit claims or this disclosure never in any form.
Example
Provide following example to illustrate, rather than limit the invention of being advocated.
Example 1
8 liters of fermentations
Described case description is used to comprise non-natural amino acid whose hGH polypeptide expression method.Host cell is to change shape through being used for the body of constructing that quadrature tRNA, quadrature aminoacyl tRNA synthetase and coding comprise the polynucleotide of the hGH polypeptide of selecting codon.
Preparation
Prepare aseptic alkali, i.e. 5.5M salt of wormwood (0.5L) and by steam or filter sterilization.The aseptic 25%v/v polyalkylene defoamer of preparation such as StruktolJ673 (0.1L) and pass through wet sterilization.Do not need acid.Concentrated charging substratum (4L, regulation) of preparation and filter sterilization are in aseptic feed chute or bioprocessing bags.
Set fermentor tank.With 3.91L alkali salt solution with its sterilization.Make fermentor tank reach following condition: temperature=37 ℃, pH=6.9,1VVM air.0.092L is concentrated the charging substratum add fermentor tank to.Add the 50mg/mL kantlex (kanamycin) of 4mL.
The solution of preparation glycerine and pectinose (yeast extract optionally) and following reagent:
Trace-metal (through wet sterilization or sterilization after filtration)
Figure A20058004446301221
Figure A20058004446301231
VITAMIN (sterilization after filtration)
Figure A20058004446301232
Glucose (through wet sterilization or sterilization after filtration)
Figure A20058004446301233
1M MgSO4 (through wet sterilization or sterilization after filtration)
Figure A20058004446301234
Ammonium sulfate, 400g/l (through wet sterilization or sterilization after filtration)
Figure A20058004446301235
5.5M K 2CO 3(through wet sterilization or sterilization after filtration)
Figure A20058004446301236
1M L-L-LEU (sterilization after filtration)
Figure A20058004446301237
1M L-L-iLeu (sterilization after filtration)
Alkali salt, 1X (through wet sterilization or sterilization after filtration)
Figure A20058004446301241
Concentrate charging
Figure A20058004446301242
The batch culture base
Figure A20058004446301243
Technology
Press the performed technology of the indicated description of table 2.
Figure A20058004446301244
The feed time table is pressed in the table 3 indicated, and the fermentation feed flow rate as shown in Figure 1.Also referring to Fig. 2.
Figure A20058004446301252
Modification to described scheme is being induced step (step IV) and is being collected step (step V) and finish.Reach about 100 to about 120 OD at culture 600After, a) inducing preceding 1.5 hours transmission glycerine pills; B) add pAF and converted yeast extract/glycerine feed in preceding 1 hour to inducing; 3) inducing preceding 0 hour interpolation pectinose; 4) finish to induce and last 8 hours.
Example 2
HGH purifying, Pegylation and hGH-PEG purification process
From colibacillary tenuigenin preparation
1. Xi Baorongxiezuoyong ﹠amp; The hGH oxidation
Making 850 gram bacterial cell bead resuspending is 25% solid mixture to obtain in the 20mM TRIS of 2550ml (3 volume), pH 8.5 dissolving damping fluids.Roughly the culture in 4 liters the fermented liquid will produce described 850 gram bacterium beads.At room temperature mixture is stirred 30-60 minute, and make suspension pass through Micro Fluid bed treater twice, simultaneously 15,000psi is cooling down.Under 4 ℃, with 13,500 * g that lysate is centrifugal in the JA10 turner, and collect supernatant liquid.The 0.1M GSSG (FW 612.6) that adds prepared fresh is so that the molar ratio of GSSG and hGH is roughly 16.To make up stirring and reach thorough mixing, and the pH value will be adjusted to 7.2-7.4 with 1M NaOH.Under 4 ℃ with the mixture stirred overnight after, water is 1.6-1.9mS/cm with its dilution up to its specific conductivity.Sample is appointed as the GHQFF filler that has lot number.
2. tubing string 1-Q sepharose FF chromatography
Column dimension is as follows: INdEX100/500,100mmI.D. * 21.5cm=1688ml.The GHQFF buffer A is had the specific conductivity of 0.5mS/cm by pH 6.5 10mM Bis-TRIS forms, and the GHQFF buffer B is had the specific conductivity of 90mS/cm by pH 6.5 1M NaCl forms.Concerning handling sample, flow rate is 90ml/min, and concerning cleaning, flow rate is 40ml/min.
Make the AKTA system source of reducing phlegm and internal heat.For making QFF tubing string reduce phlegm and internal heat source and balance, use " QFF reduce phlegm and internal heat source balance " program: the 1M NaOH/1M NaCl with the MilliQ water of 2 column volumes, 2 column volumes washs tubing string, cultivated 30 minutes, with the GHQFF buffer B washing of 3 column volumes, use the GHQFF buffer A balance of 4 column volumes then.
Sample GHQFF filler is loaded on the anion-exchange column.With the GHQFF buffer A of 5 column volumes washing tubing string, and with the 6%GHQFF buffer B elution in A of 4 column volumes.Collect primary front.Begin sample collection and finish with roughly 0.85mS/cm and 166mAU with 220mAU roughly.To be appointed as the GHQFF pond that has lot number through the eluant of collecting, and its color is a brown orange.Under 4 ℃, the pond is stored whole night.3 batches average step productive rate is 84.7%.
GHQFF buffer B washing tubing string with 2-3 column volume.1M NaOH/1MNaCl with 2 column volumes sucks with pump, and tubing string was cultivated 1-6 days.If in 6 days, do not use tubing string, with the MilliQ water of the buffer B of the 1M NaOH/1M NaCl of 1 column volume, 3 column volumes, 2 column volumes and the 20%EtOH of 2.5 column volumes it is washed so.
The large-scale cleaning of a tubing string is carried out in every 3-5 circulation.After 1M NaOH/1M NaCl cultivates, carry out following steps: with the upwards mobile washing of the Q tubing string cleaning buffer of 2.5 column volumes, cultivated 60-80 hour, with 0 20%EtOH washing of the MilliQ water of 1.5 column volumes, 1 column volume to 70%EtOH, the 70%EtOH of 5 column volumes, 2.5 column volumes.Q tubing string cleaning buffer is made up of 0.5%Triton X-100,0.1M acetate.
(3.UF/DF ultrafiltration/saturating filter) I
Following strainer is used for described program: Sartorius Sartocon Slice 10K Hydrosart box, 1000cm 2GHQFF pond sample concentration is tapered to about 450ml (or in the retention flask, about 200ml).Then, with pH 6.3 by 10mM Bis-TRIS, 1mM MgCl 2The GHCHT buffer A of the 2.7L (6-volume) that forms is filtered thoroughly.Collect after the retention, with the damping fluid rinse-system of 300ml and with washing fluid and retention combination.4,000rpm (under 2,862 * g) with centrifugal 5 minutes of retention, and collects supernatant liquid.Supernatant liquid is appointed as the GHCHT filler that has lot number.Described sample is to handle in 2 hours or store whole night down at 4 ℃.
4. tubing string 2-pottery hydroxylapatite (CHT) chromatography (I type CHT, 40 μ m)
Column dimension is as follows: INdEX100/500,100mmI.D. * 10.5cm=824ml.The GHCHT buffer A is by 10mM Bis-TRIS, 1mM MgCl 2Form, pH 6.3, have the specific conductivity of 0.94mS/cm.The GHCHT buffer B is by 10mM Bis-TRIS, 0.5M MgCl 2Form, pH 6.3, have the specific conductivity of 80.5mS/cm.Concerning handling, flow rate is 90ml/min, and concerning cleaning, flow rate is 40ml/min.
Make the AKTA system source of reducing phlegm and internal heat.For making CHT tubing string reduce phlegm and internal heat source and balance, operation " CHT reduce phlegm and internal heat source balance " program: the 1M NaOH/1M NaCl with the MilliQ water of 2 column volumes, 2 column volumes washs the CHT tubing string, cultivates 30 minutes, with the 0.5M NaPO of 3 column volumes 4/ pH 7.0 washs, and uses the GHCHT buffer A balance of 4 column volumes then.Then the GHCHT packing samples is loaded on the tubing string.GHCHT buffer A washing tubing string with 5 column volumes.
Carry out elution with the linear gradient of the 0-40%GHCHT buffer B that surpasses 5 column volumes, the stepping gradient that surpasses 3 column volume 40%GHCHT buffer B, and wash with the 100%GHCHT buffer B that surpasses 2 column volumes.Collect main peak.Begin to collect with roughly 26mAU, 20mS/cm, 28%GHCHT buffer B, and so that roughly 86mAU, 34mS/cm, 40%GHCHT buffer B finish.Collected eluant is appointed as the GHCHT pond that has lot number.Under 4 ℃, the pond is stored whole night.3 batches average step productive rate is 96.3%.
0.5M NaPO with 3 column volumes 4/ pH 7.0 washing CHT tubing strings.Tubing string is stayed in the described phosphate buffered saline buffer, or carried out following steps: with the 1M NaOH/1M NaCl of 2 column volumes, the 0.5MNaPO of 3 column volumes 4The 20%EtOH of 7.0,2.5 column volume MilliQ water of/pH and 2.5 column volumes upwards flows and washs tubing string.
5. tubing string 3-phenyl sepharose gel HP chromatography
Column dimension is as follows: INdEX100/500,100mmI.D. * 9.7cm=761ml.The GHPhe buffer A is by 20mMNaPO 4, 2M NaCl forms, pH 7.0, have the specific conductivity of 163mS/cm, and the GHPhe buffer B is by 20mM NaPO 4Form, pH 7.0, have the specific conductivity of 3.2mS/cm.Concerning handling sample, flow rate is 90ml/min, and concerning cleaning, flow rate is 40ml/min.
Make the AKTA system source of reducing phlegm and internal heat.For making Phe tubing string reduce phlegm and internal heat source and balance, operation " PheHP reduce phlegm and internal heat source balance " program: the 1M NaOH/1M NaCl with the MilliQ water of 2 column volumes, 2 column volumes washs tubing string, cultivates 30 minutes, uses the GHPhe buffer A balance of 4 column volumes then.
Add solid NaCl to the GHCHT pond and reach 2M.At room temperature mixture is stirred and reached dissolving in 1-2 hour, and make solution be warmed up to roughly 20 ℃.Be the amount (Zg) of calculating required NaCl: (V+Z/4000) * 2 * 58.44=Z, or Z=116.88V/ (1-116.88/4000), wherein V is the volume in the GHCHT pond of liter.
GHCHT pond+NaCl mixture is loaded on the tubing string.GHPhe buffer A washing tubing string with 3 column volumes.Carry out elution with following complex gradient: surpass 10% stepping of 3 column volumes the GHPhe buffer B, surpass 7 column volumes 10-80%GHPhe buffer B gradient, surpass the 80%GHPhe buffer B stepping gradient of 2 column volumes and surpass the 100%GHPhe buffer B stepping gradient of 3 column volumes.Collect main peak.Begin to collect and so that roughly 43mAU, 54mS/cm, 80%GHPhe buffer B finish with roughly 17.3mAU, 111mS/cm, 46.7%GHPhe buffer B.Collected eluant is appointed as the GHPhe pond that has lot number and it is a colourless solution.In 2 hours, carry out next procedure, or under 4 ℃, the pond is stored whole night.3 batches average step productive rate is 94.6%.
With the upwards mobile washing Phe of the 1M NaOH tubing string of 2 column volumes, cultivate 30min, wash with the MilliQ water of the GHPhe buffer A of 3 column volumes, 3 column volumes and the 20%EtOH of 2.5 column volumes.After 3-5 the circulation, the upwards mobile washing Phe of 1M NaOH tubing string with 2 column volumes, cultivate 30min, with the 70%EtOH washing of the MilliQ water of the GHPhe buffer A of 3 column volumes, 3 column volumes, the 0-70%EtOH that surpasses 1 column volume, 3 column volumes, and be stored among the 20%EtOH.
(6.UF/DF ultrafiltration/saturating filter) II
Following strainer is used for described program: Sartorius Sartocon Slice 10K Hydrosart box, 1000cm 2The GHPhe pond is tapered to about 450ml (or in the retention flask, about 200ml).The GH prescription damping fluid of using 2.7L (6-volume) then is with its saturating filter, and described GH prescription damping fluid is made up of 20mM Trisodium Citrate, 20g/L glycine, 5g/L mannitol, and pH 6.0.Sample concentration is tapered to about 360ml.Collect retention.With the GH prescription damping fluid rinse-system of 300ml and with washing fluid and retention combination.4,000rpm (under 2,862 * g) with centrifugal 5 minutes of retention, and collects supernatant liquid.Supernatant liquid is appointed as Y35pAF-cBx, and is also referred to as " main body in the processing (in-process bulk) ".With the main body five equilibrium in handling and-80 ℃ of storages down.
The ultimate production of Y35pAF is every liter of fermented liquid 435mg.To analyze based on 3 kinds of HPLC methods (RP-HPLC, SEC-HPLC, IEX-HPLC) and SDS-PAGE, purity is＞90%.
(7.UF/DF ultrafiltration/saturating filter) IIa
Following thickener/strainer is used for described program: the Amicon Stirred Cell (200ml) with YM10 film (63.5mm).Reaction buffer is made up of 20mM sodium acetate, 20g/L glycine, 5g/L mannitol, 1mM EDTA, and pH 4.0.Main body during use is handled from the part of step 6, such as the Y35pAF of 250mg, and 10% acetate by interpolation 10-12% (v/v) is adjusted to roughly 4 with the pH value.Sample concentration is tapered to 25-50ml, and the interpolation reaction buffer reaches roughly 180ml.Re-treatment is up to reaching altogether＞500 times buffer exchange.Sample concentration is arrived roughly 25ml.Collect retention, and under 2,000 * g centrifugal 3 minutes to remove any throw out.Supernatant liquid is appointed as the Y35pAF-cBx/pH4 that has the date.
The protein concn of Y35pAF-cBx/pH4 is by measuring the A through the sample of 20 times of dilutions 276, use A 276 1 Mg/ml=0.818 determines.Arrive 8mg/ml by the concentration adjustment of diluting Y35pAF-cBx/pH4 with reaction buffer.
8. pegylation reaction
Use the molar ratio of PEG: Y35pAF=10 to calculate the required amount of 30K MPEG-oxyamine.PEG powder art is weighed and at room temperature slowly add in the 8mg/ml Y35pAF solution, and add the back in each time and mix with spatula.Reaction mixture is placed under 28 ℃, shook gently 18-48 hour simultaneously.Confirm Pegylation by the sds gel method.Be reflected at and form the oxime key between hGH and the PEG.
9. tubing string 4-source electrode Q chromatography (Source Q Chromatography) (30 μ m)
Column dimension is as follows: XK26/20,26mm I.D. * 17cm=90ml.Source electrode Q buffer A is made up of 10mM TRIS, and pH 7.0, has the specific conductivity of 0.9mS/cm.Source electrode Q buffer B is made up of 10mM TRIS, 1M NaCl, and pH 7.0, has the specific conductivity of 93mS/cm.Flow rate is 6ml/min.
Make the AKTA system source of reducing phlegm and internal heat.For making source electrode Q tubing string reduce phlegm and internal heat source and balance, operation " source electrode Q reduce phlegm and internal heat source balance " program: the 1M NaOH/1M NaCl with the MilliQ water of 2 column volumes, 2 column volumes washs source electrode Q tubing string, cultivated 30 minutes, with the source electrode Q buffer B washing of 5 column volumes, use the source electrode Q buffer A balance of 5 column volumes then.
Add the 0.5M TRIS alkali of 20% (v/v) to reaction mixture from step 8.Carry out 20 times of dilutions with the source electrode Q buffer A of 9-volume and the MilliQ water of 10-volume.Then mixture is loaded on the tubing string.Source electrode Q buffer A washing tubing string with 5 column volumes.Linear gradient with the 0-10% source electrode Q buffer B that surpasses 20 column volumes is carried out elution.Collect first primary front.Collected eluant is appointed as the source electrode Q pond that has lot number.Under 4 ℃, the pond is stored whole night.
(10.UF/DF ultrafiltration/saturating filter) III
Following thickener/strainer is used for described program: the Amicon Stirred Cell (200ml) with YM10 film (63.5mm).The WHO damping fluid is by 2.5g/L NaHCO 3, 20g/L glycine, 2g/L mannitol, 2g/L lactose form, pH 7.3.
Source electrode Q pond is concentrated to 20-30ml, and interpolation WHO damping fluid reaches roughly 180ml.Re-treatment is up to reaching altogether＞600 times buffer exchange.Then sample concentration is arrived 2mg/ml or desired concentration.Collect retention, and in stink cupboard with 0.2 μ m membrane filtration sterilization.Aseptic sample is appointed as the PEG30-cY35pAF that has lot number.
The equivalent hGH concentration of PEG30-cY35pAF is by measuring the A of diluted sample 276, by using A 276 1mg/ml=0.818 determines, carries out three dilutions and measurement.From the overall yield of step 7 is roughly 20%.To analyze based on HPLC and SDS-PAGE, PEG-Y35pAF purity is＞95%.
Example 3
HGH purifying, Pegylation and hGH-PEG purification process
From colibacillary pericentral siphon preparation
1.hGH pericentral siphon discharge
The 800 gram bacterial cell bead resuspending that will obtain from 4 liters of fermented liquids roughly in the 4-6 ℃ of PR damping fluid of 3200ml (4-volume) (50mM TRIS, 2mM EDTA, 0.07%Triton X-100, pH 8.0; In the specific conductivity=3mS/cm) to obtain 20% solid.Under 4-6 ℃ the suspension stirring after 1 hour, is being added the final urea concentration of the 8M urea of 150ml with acquisition 0.3M.Then, under 4-6 ℃, described suspension was stirred 1 hour.Under 4 ℃, in J20 turner (Avanti J20XP whizzer-Beckman Coulter), under 15,000 * g with centrifugal 25 minutes of suspension.Collect supernatant liquid, measure its volume (roughly 3.4L).Sample is appointed as the PRS that has date and lot number.
(2.UF/DF ultrafiltration/saturating filter) I
Following strainer is used for described program: Sartorius Sartocon Slice 10K Hydrosart box, 1000cm 2Other parameters comprise: the filtrate flows speed of 80 ml/min and the TMP of 14psi roughly.
, and allowed circulation 30-45 minute the system source of reducing phlegm and internal heat with 1N NaOH.Drop to below 8 up to pH with 2 liters MilliQ water rinse-system roughly.Balance is to last at least 5 minutes with QFF buffer A (10mM Bis-TRIS, pH 6.5) to finish.PRS is tapered to roughly 1.6 liters (or in the retention container, roughly 1.4 liters).The QFF buffer A of using 5-volume (about 7 liters) then is with its saturating filter.Collect after the retention, with the damping fluid rinse-system of 300ml and with washing fluid and retention combination.The sample that is made up is appointed as the QFF filler that has lot number.It is a brown.In 2 hours, handle described sample or it is descended storage whole night at 4 ℃.
With MilliQ water rinse-system and by circulation 30-45 minute, with 1N NaOH cleaning.Then, with MilliQ water finish flushing up to the pH value less than 8.Box is stored among the 0.1N NaOH.
3. tubing string 1-Q sepharose FF chromatography
Column dimension is as follows: 50mm I.D. * 6.3cm=123ml (XK26/20 tubing string).Flow rate is 35ml/min.The QFF buffer A is made up of 10mM Bis-TRIS, and pH 6.5, has the specific conductivity of 0.6mS/cm.High-salt buffer is made up of 10mM TRIS, 2M NaCl, and pH 7.0, has the specific conductivity of 156mS/cm.The QFF buffer B is made up of 10mM Bis-TRIS, 0.1M NaCl, and pH 6.5, has the specific conductivity of 11.5mS/cm.
Make the AKTA system source of reducing phlegm and internal heat.For finishing described processing, operation " AKTA reduce phlegm and internal heat source " program 3 times: in first time during working procedure, whole damping fluid pipelines are placed in the MilliQ water, are placed among the 1NNaOH when moving for the second time then.Last 30 minutes and finish cultivation, and when moving for the third time, once more the damping fluid pipeline is placed in the MilliQ water.Operation " QFF reduce phlegm and internal heat source balance " program is source and balance so that the QFF tubing string reduces phlegm and internal heat: with the MilliQH of 2 column volumes 2The 1N NaOH/1M NaCl washing QFF tubing string of O, 2 column volumes is cultivated 30min, with the high-salt buffer washing of 3 column volumes, uses the QFF buffer A balance of 4 column volumes then.
Then the QFF filler is loaded on the tubing string.With the QFF buffer A of 5 column volumes and the 15%QFF buffer B washing tubing string in A of 5.5 column volumes.The 60%QFF buffer B in A with 4.5 column volumes is carried out elution, and collects the elution peak.Collected eluant is appointed as the QFF pond that has lot number, and it is faint yellow.Under 4 ℃, the pond is stored whole night.
High-salt buffer washing tubing string with 3 column volumes.Then, the 1N NaOH/1MNaCl with 3 column volumes sucks with pump, and lasts 1-6 days and cultivate.If in 6 days, do not use tubing string, use the high-salt buffer of the 1N NaOH/1M NaCl of 1 column volume, 3 column volumes, the MilliQ H of 3 column volumes so 2The 20%EtOH of O and 2.5 column volumes or 10mM NaOH wash it.Every 3-5 circulation carried out the cleaning on a large scale of a tubing string so that after 1N NaOH/1M NaCl cultivates, Q tubing string cleaning buffer (0.5%Triton X-100,0.1M acetate) with 3 column volumes upwards flows its washing, cultivated 60-80 hour, with the MilliQ H of 1.5 column volumes 20 to 70%EtOH of O, 1 column volume, the 20%EtOH washing of the 70%EtOH of 5 column volumes and 2.5 column volumes.
4. tubing string 2-phenyl sepharose gel HP chromatography
Column dimension is as follows: 50mm I.D. * 7.5cm=147ml (XK26/20 tubing string).Flow rate is 35 ml/min.The Phe buffer A is made up of 10mM TRIS, 2M NaCl, and pH 7.0, has the specific conductivity of 156mS/cm.The Phe buffer B is made up of 10mM TRIS, and pH 7.0, has the specific conductivity of 0.9mS/cm.
Make the AKTA system source of reducing phlegm and internal heat.Operation " AKTA reduce phlegm and internal heat source " program 3 times: in first time during working procedure, whole damping fluid pipelines are placed in the MilliQ water, are placed among the 1N NaOH when moving for the second time then.Last 30 minutes and finish cultivation, when moving for the third time, once more all damping fluid pipelines are placed in the MilliQ water then.Operation " PheHP reduce phlegm and internal heat source balance " program is source and balance so that the Phe tubing string reduces phlegm and internal heat: with the MilliQ H of 2 column volumes 2The 1M NaOH/1M NaCl washing tubing string of O, 2 column volumes is cultivated 30min, uses the Phe buffer A balance of 4 column volumes then.
Add solid NaCl to the QFF pond and reach 2M.At room temperature mixture is stirred 1-2 hour with dissolving NaCl, and solution is warmed up to roughly 20 ℃.Calculate the amount (Zg) of required NaCl: (V+Z/4000) * 2 * 58.44=Z, or Z=116.88V/ (1-116.88/4000), wherein V is the volume in the QFF pond of liter.
QFF pond+NaCl is loaded on the tubing string.Phe buffer A washing tubing string with 5 column volumes.Carry out elution with following complex gradient: surpass 10 column volumes the 0-45%B linear gradient, surpass the 45%B stepping gradient of 2 column volumes and surpass the 100%B stepping gradient of 3 column volumes.During gradient elution, collect main peak.Collected eluant is appointed as the Phe pond that has lot number, and it is a colourless solution.Carry out next step or the pond is stored at 4 ℃.
With the upwards mobile washing Phe of the 1M NaOH tubing string of 2 column volumes, cultivate 30min, with the Phe buffer A of 3 column volumes, the H of 3 column volumes 2The 20%EtOH of O and 2.5 column volumes or 10mM NaOH washing.After 3-5 the circulation,, cultivate 30min, with the GH Phe buffer A of 3 column volumes, the H of 3 column volumes with the upwards mobile washing Phe of the 1M NaOH tubing string of 2 column volumes 2The 70%EtOH washing of O, the 0-70%EtOH that surpasses 1 column volume, 3 column volumes, and finally be stored among the 20%EtOH.
(5.UF/DF ultrafiltration/saturating filter) II
Following strainer is used for described program: Sartorius Sartocon Slice 10K Hydrosart box, 200cm 2Other parameters comprise: the filtrate flows speed of 15ml/min and the TMP of 14psi.Preliminary prescription damping fluid is made up of 20mM Trisodium Citrate, 20g/L glycine, 5g/L mannitol, and pH 6.0, have the specific conductivity of 4.7mS/cm.
, and allowed circulation 30-45 minute the system source of reducing phlegm and internal heat with 1N NaOH.Drop to below 8 up to the pH value with 2 liters MilliQ water rinse-system roughly.Finish balance with preliminary prescription damping fluid and last at least 5 minutes.
GH Phe pond tapered to make an appointment with the people to cause 350ml (or in the retention flask, roughly 200ml).Preliminary prescription damping fluid with 2.1 liters (6-volumes) is finished filter.Then, sample concentration is tapered to roughly 350ml, and collect retention.With the damping fluid rinse-system of 300ml and with washing fluid and retention combination.4,000rpm (under 2,862 * g) with centrifugal 5 minutes of retention, and collects supernatant liquid.Supernatant liquid is appointed as Y35pAF-pBx, and is also referred to as " main body in the processing ".
The protein concn of Y35pAF-pBx is by measuring the A of diluted sample 276, use A 276 1mg/ml=0.818 determines.Can be in the main body in 4 ℃ of following stores processor.For carrying out prolonged storage, with its five equilibrium and remain on-80 ℃.
With MilliQ water rinse-system and by circulation 30-45 minute, clean with 1N NaOH.Then, with MilliQ water it is washed up to the pH value below 8.Box is stored among the 0.1N NaOH.
(6.UF/DF ultrafiltration/saturating filter) IIa
Use following thickener/strainer: Amicon Stirred Cell (350ml) with YM10 film (76mm).Reaction buffer is made up of following material: 20mM sodium acetate, 20g/L glycine, 5g/L mannitol, 1mM EDTA, pH 4.0, have the specific conductivity of 2.6mS/cm.
With Pyroclean with the system source of reducing phlegm and internal heat.In Pyroclean, all components was cultivated 30 minutes.Finish and use flushing that MilliQ water carries out up to A 205Less than 0.01.
By adding 10% acetate of 10-12% (v/v), the pH value of the main body (such as 300mg) during a part handled is adjusted to roughly 4.Described sample concentration is tapered to 25-50ml, and the interpolation reaction buffer reaches roughly 350ml.Re-treatment is up to reaching altogether＞500 times buffer exchange.Then, sample concentration is arrived roughly 30ml.Collect retention, and under 2,000 * g centrifugal 3 minutes to remove any throw out.Supernatant liquid is appointed as the Y35pAF-pBx/pH4 that has the date.For carrying out prolonged storage, with its five equilibrium and remain on-80 ℃.
The protein concn of Y35pAF-pBx/pH4 is by measuring the A through the sample of 20 times of dilutions 276, by using A 276 1mg/ml=0.818 determines.By diluting with reaction buffer, with the concentration adjustment of Y35pAF-pBx/pH4 to 8mg/ml.
7. pegylation reaction
Use the molar ratio of PEG: Y35pAF=5 to calculate the required amount of 30K MPEG-oxyamine.With the PEG powder weighing and at room temperature slowly add 8mg/ml Y35pAF-pBx/pH4 solution to, stir simultaneously.Under 28 ℃, under stirring gently, reaction mixture was placed 39-50 hour.Confirm Pegylation by carrying out SDS-PAGE.Be reflected at and form the oxime key between hGH and the PEG.
8. tubing string 3-source electrode Q chromatography (30 μ m)
Column dimension is as follows: XK26/20,26mm I.D. * 17cm=90ml.Flow rate is 8 ml/min.Source electrode Q buffer A is made up of 10mM TRIS, and pH 7.0, has the specific conductivity of 0.9mS/cm.Source electrode Q buffer B is made up of 10mM TRIS, 1M NaCl, and pH 7.0, has the specific conductivity of 87mS/cm.
Be source that the AKTA system is reduced phlegm and internal heat, operation " AKTA reduce phlegm and internal heat source " program 3 times:, whole damping fluid pipelines are placed in the MilliQ water, and when moving for the second time, are placed among the 1N NaOH in first time during working procedure.Last 30 minutes and finish cultivation, and when moving for the third time, once more all damping fluid pipelines are placed in the MilliQ water.For making source electrode Q tubing string reduce phlegm and internal heat source and balance, operation " source electrode Q reduce phlegm and internal heat source balance " program: with the MilliQ H of 2 column volumes 2The 1M NaOH/1M NaCl washing source electrode Q tubing string of O, 2 column volumes was cultivated 30 minutes, with the source electrode Q buffer B washing of 5 column volumes, used the source electrode Q buffer A balance of 5 column volumes then.
Add the 0.5M TRIS alkali of 20% (v/v) to reaction mixture from previous steps.With the source electrode Q buffer A of 9-volume and the MilliQ H of 10-volume 2O carries out 20 times of dilutions.Make diluted material by 0.45 μ m strainer.Then filtrate is loaded on the tubing string.Source electrode Q buffer A washing tubing string with 5 column volumes.Linear gradient with the 0-10% source electrode Q buffer B that surpasses 20 column volumes is carried out elution.Frac-950 is used for 13 milliliters/elution fraction collection cut.Operation SDS-PAGE is with cell on first primary front.The cut that is compiled is appointed as the source electrode Q pond that has lot number.Under 4 ℃, the pond is stored whole night.
(9.UF/DF ultrafiltration/saturating filter) III
Use following thickener/strainer: Amicon Stirred Cell (350ml) with YM10 film (76mm).Preliminary prescription damping fluid is made up of 20mM Trisodium Citrate, 20g/L glycine, 5g/L mannitol, and pH 6.0, have the specific conductivity of 4.7mS/cm.
Make the system source of reducing phlegm and internal heat with Pyroclean.In Pyroclean, all components was cultivated 30 minutes.Finish and use flushing that MilliQ water carries out up to A 205＜0.01.
Source electrode Q pond is concentrated to 20-40ml, and the preliminary prescription of interpolation damping fluid reaches roughly 350ml.Re-treatment is up to reaching altogether＞600 times buffer exchange.Sample concentration is arrived 2mg/ml or desired concentration.Collect retention, and in stink cupboard with 0.2 μ m membrane filtration sterilization.Aseptic sample is appointed as the PEG30-pY35pAF that has lot number.
The equivalent hGH concentration of PEG30-pY35pAF is by measuring the A of diluted sample 276, by using A 276 1 Mg/ml=0.818 determines, and carries out three dilutions and measurement.Can store PEG30-pY35pAF down at 4 ℃.For carrying out prolonged storage, with its five equilibrium and remain on-80 ℃.
Used the bacterial strain of disallowable DH10B of araB gene (fis) and W3110 to finish the pericentral siphon delivery formulations.Two kinds of bacterial strains are to construct body through quadrature tRNA, quadrature aminoacyl tRNA synthetase and hGH to change shape.Analyze PEG-Y35pAF purity＞95% based on HPLC and SDS-PAGE.
Example 4
Compare the hGH preparation: pericentral siphon discharges contrast tenuigenin (homogenizing)
The SDS-PAGE that Fig. 3, plate A and B are illustrated in the hGH that produces in the intestinal bacteria analyzes.Make pericentral siphon and discharge a batch (fermentation crowd 050425B2; The cell masses of 800 grams) and tenuigenin batch (dissolve by the Micro Fluid bed; Fermentation crowd 050414B1; The cell mass of 60 grams).On 123ml Q FF tubing string, use by 10mM Bis-TRIS and form, the QFF buffer A of pH 6.5 and by 10mM Bis-TRIS, pH 6.5, and the QFF buffer B that 0.1M NaCl forms is moved each batch.During elution, carry out 3 kinds of cuts: 15,60 and 100% buffer B (being respectively 15mM NaCl, 60mM NaCl and 100mM NaCl).Analyze from isolating aliquots containig by SDS-PAGE.The swimming lane of plate A and B is as follows: swimming lane 1=WHO hGH standard; Swimming lane 2=filler; Swimming lane 3=BE/FT; Swimming lane 4=15% buffer B; Swimming lane 5=60% buffer B; With swimming lane 6=100% buffer B.
Example 5
5 liters of fermentation process
Described case description is used to comprise non-natural amino acid whose hGH polypeptide expression method.The bacterial strain of employed host cell is modified W3110 clone.Host cell is to change shape through being used for the body of constructing that quadrature tRNA, quadrature aminoacyl tRNA synthetase and coding comprise the polynucleotide of the hGH polypeptide of selecting codon.Technical process as shown in Figure 4.
Preparation
Prepare following reagent:
Trace elements (through wet sterilization)
Figure A20058004446301341
Figure A20058004446301351
VITAMIN (sterilization after filtration)
1M MgSO 4(through wet sterilization)
Figure A20058004446301353
Be used for all ammonium hydroxide, 15% is NH 3 *
Figure A20058004446301354
Be used for all alkali salts, 1X (through wet sterilization)
Concentrate charging (the sterility blended is through germ-resistant component)
Figure A20058004446301356
*: behind steam sterilization, add.
The batch culture base
Figure A20058004446301357
Figure A20058004446301361
*: behind steam sterilization, add.
Consume yeast extract/glycerol mixture (through wet sterilization)
Figure A20058004446301362
Be used for all kantlex raw materials (sterilization after filtration)
Figure A20058004446301363
Use the same day, preparing following reagent:
Be used for all to acetyl phenyl alanine (pAF) *(sterilization after filtration)
Figure A20058004446301364
*: final volume becomes 21.25ml.Gained solution after using all to filter.
Be used for all L-(+)-pectinose 20% (sterilization after filtration)
Figure A20058004446301365
*: the gained solution after using all to filter.
Concerning each fermentation, carry out following steps.Preparation 25%Struktol J 673 (0.1L) and wet sterilizations.Preparation 15%NH 3* H 2O (0.3L) is used for pH value control and as nitrogenous source.Preparation 10%H 3PO 4(0.2L) be used for the control of pH value.Preparation concentrates charging, 1L in feed containers 1.Preparation YE/ glycerol mixture respectively is 2L in feed containers 2.
Set up fermentor tank.With 2.5L alkali salt solution with its sterilization.Make fermentor tank reach following condition: temperature=37 ℃, pH=6.9, based on the 1.0VVM air (air-flow can increase up to 2VVM) of 5L working volume.
Add concentrated charging to fermentor tank, and add the 50mg/mL kantlex of 2.5ml.
Technical process
The 1st day (Phase I):
The about 1ul of picking from the intestinal bacteria MCB (master cell bank, glycerine raw material), and the glycerine raw material of 1 μ l transferred in 2ml batch culture base+kantlex in the culture tube.The composition of batch culture base as mentioned above.
The 2nd day (Phase and III):
Phase:
Culture tube contains the 1-6 (OD that has an appointment 600) cell density.The culture tube culture of 0.01-2ml is transferred to 250ml to be shaken in 60ml batch culture base+kantlex in the bottle.Only use the healthy cell that is not subject to any carbon hunger.The composition of batch culture base as mentioned above.
Phase I:
The inoculation fermentation jar reaches about 0.05 initial OD 600The amount (in liter) of required cell from Phase is
2 . 5 L × 0.05 4 = 0.031 L If (flask OD 600=4)
Make cell pursue batch growth roughly 10 hours.Specified time is to stipulate through the loss of culture by glycerine.The glycerine loss is the unexpected reduction by STIRR speed, then pO 2The increase of signal is indicated.Beginning is through spissated glycerine feed.Feeding rate is based on formula I:
F ( 0 ) = μ set Y x / s × X ( 0 ) × V ( 0 ) × Exp ( μ set × 0 ) × 1 S f
μ set＝0.15h -1；
X(0)＝Yx/s*8.0g/l
V(0)＝2.51；
Exp(μ set*0)＝1；
S f＝400g/l
With above-mentioned value substitution equation, F (0)=7.5ml/h.The usage ratio factor will be so true F (0) ' will be 8.6ml/h.
F(t)′＝F(0)′*Exp(μ set*t)。Concerning t=13 hour, F (t) '=60.4 milliliter/hour.
Flow rate is remained on 60.4 milliliters/hour last 1 hour.
Concerning operation, when charging 2 beginnings, add pAF.The pectinose inductor is to add interpolation in back 1 hour at pAF.Keep final charging 2 flow rates up to collection.
The 3rd day (Phase IV and V)
Phase IV:
Cultivate OD 600Reach about 50 to 60.Before inducing 1 hour (feed time=14 hour), 1) stops to concentrate charging (in this time, speed=60.4 milliliter/hour).2) with 108.7 milliliters of/hour beginning YE/ glycerine (every liter 200g YE and 170g glycerine) charging.3) add the 21.25ml pill that contains 4.0g pAF.PH 6.9 times, continue control pH value, use 15% ammonium hydroxide and 10% phosphoric acid to regulate the pH value in case of necessity.3a) charging 2 speed are increased linearly and last 3 hours, reach 141.3 milliliters/hour during in feed time=17 hour.Culture OUR should remain on about 250mmol/1/ hour.3b) carbon source being changed continued 1 hour.3c) cell (the OD of preservation q.s 600* ml=2) being used for SDS-PAGE analyzes.
When inducing (feed time=15 hour), induce with 1.25ml 20% (w/v or 200g/l) L-(+)-pectinose.At the cell (OD that induced back 4 hours, 6 hours and preserved in 8 hours q.s 600* ml=2) being used for SDS-PAGE analyzes.Make inducing sustained 8 hours.
Stage V:
When inducing end, check and cultivate OD 600Preserve the cell (OD of q.s 600* ml=2) being used for SDS-PAGE analyzes.Be used for evaluation by 2 * 200 milliliters of cultures of centrifugal collection by ELISA.15, under the 000g, use bucket to come collecting cell in centrifugal 22 minutes, and under-80 ℃ with cell freezing.
Described program has been expanded in proportion to 100 liters of cultures.
Example 6
HGH purifying, Pegylation and hGH-PEG purification process
From colibacillary pericentral siphon preparation
1.hGH pericentral siphon discharge
With 1.9 kilograms of bacterial cell mass resuspending in 4 ℃ of PR damping fluids (50mM TRIS, 10mM EDTA, 0.07%Triton X-100, pH 8.0) of roughly 7.6 liters (4-volumes) to obtain 20% solid.Under 4 ℃ the suspension stirring after 1 hour, is being added 8M urea to obtain the final urea concentration of 0.3M.In 48 hours of preparation, use the 8M urea soln.Under 4 ℃, described suspension was stirred 1 hour then.Under 4 ℃, in fixed angle J20 turner (Avanti J20XP whizzer-Beckman Coulter), under 15,000 * g with centrifugal 45 minutes of suspension.Collect supernatant liquid, and measure its volume (roughly 7.7L).Sample is appointed as the PRS that has date and lot number.
Make PRS filter prefilter, Sartopure GF21.2 μ m container (1000cm 2) (parts #5571303P800B).When pump was set 1 (MasterFlex I/P 7529-10 type), filtrate flows speed was 0.86L/min.Collect filtrate and be appointed as the PRSF that has date and lot number.Measure the volume of PRSF.
Make PRSF filter Sartopore 20.8+0.45 μ m filtration vessel (500cm 2) (parts #5441306G700B).Collect filtrate and be appointed as the PRSFF that has date and lot number.Measure the volume of PRSFF.
Analysis in the processing comprises, confirms to compare with reference standard, and the irreducibility SDS-PAGE of existence that is the hGH of correct form analyzes A 276Measurement and be used for quantitative ELISA.
The buffer exchange of (2.UF/DF ultrafiltration/saturating filter) I-QFF
Following strainer is used for described program: Sartorius Sartocon Slice 10K Hydrosart box, 2 * 1000cm 2Other parameters comprise: the feed pressure of the filtrate of 100-160 ml/min (penetrant) flow rate, 24-26psi and the retention pressure of 5-6psi.
, and allowed circulation 30-45 minute the system source of reducing phlegm and internal heat with 1N NaOH.Drop to below 8 up to the pH value with 4 liters MilliQ water rinse-system roughly.Balance is to last at least 5 minutes with QFF buffer A (10mM Bis-TRIS, pH 6.5) to finish.PRSFF is tapered to 1/10th of its volume roughly.The QFF buffer A of using the 8-volume then is with its saturating filter.Made retention recirculation 3-5 minute.Collect after the retention, with the QFF buffer A rinse-system of 300-350 milliliter and with washing fluid and retention combination.Make sample filter Sartopore 20.8+0.45 μ m container (500cm through combination 2) (parts #5441306G700B), and collected filtrate being appointed as had the QFF filler of date and lot number.It is a brown.Measure the volume of QFF filler, and in 2 hours, handle the QFF filler or descend storage whole night at 4 ℃.
With MilliQ water rinse-system and by circulation 30-45 minute, with 1N NaOH cleaning.Then, with MilliQ water finish flushing up to the pH value less than 8.Box is stored among the 0.1N NaOH.
Analysis in the processing comprise quantitative gross protein and be identified for next step the QFF filler amount A 276Measurement, ELISA, LAL and irreducibility SDS-PAGE analyze.
3. tubing string 1-Q sepharose FF chromatography
The mobile fast post (Fast Flow) of Q sepharose is to obtain from GE Healthcare.Column dimension is as follows: 70mmI.D. * 16cm=616ml (INdEX70/500 tubing string).Throughput is that (140-160mg is based on A for every ml QFF 150mg gross protein 276) or 10mg GH (based on ELISA).Flow rate is a 100ml/min (linear speed: 156cm/h).The QFF buffer A is made up of 10mM Bis-TRIS, and pH 6.5, has the specific conductivity of 0.6mS/cm.The QFF buffer B is made up of 10mM Bis-TRIS, 0.1M NaCl, and pH 6.5, has the specific conductivity of 11.5mS/cm.
Make the AKTA detector system source of reducing phlegm and internal heat.For finishing the described source of reducing phlegm and internal heat, operation " AKTA reduce phlegm and internal heat source " program 3 times: in first time during working procedure, whole damping fluid pipelines are placed in the MilliQ water, are placed among the 1N NaOH when moving for the second time then.Last 30 minutes and finish cultivation, and when moving for the third time, once more the damping fluid pipeline is placed in the MilliQ water.With the linear speed of 30cm/h, operation " QFF reduce phlegm and internal heat source balance " program is source and balance so that the QFF tubing string reduces phlegm and internal heat: with the MilliQ H of 2 column volumes 2The 1N NaOH/1M NaCl washing QFF tubing string of O, 2 column volumes is cultivated 30min, with Q damping fluid C (10mM TRIS, 2M NaCl, pH 7.0, have the specific conductivity of the 156mS/cm) washing of 3 column volumes, uses the QFF buffer A balance of 4 column volumes then.
Then the QFF filler is loaded on the tubing string.With the QFF buffer A of 4 column volumes and the 10%QFF buffer B washing tubing string in A of 7 column volumes.The 60%QFF buffer B in A with 6 column volumes is carried out elution.Tubing string can wash with the QFF buffer B of 3 column volumes.Collect the elution peak.Collected eluant is appointed as the QFF pond that has date and lot number.In 2 hours treating pond or at 4 ℃ down storage is whole night.
Q damping fluid C washing tubing string with 3 column volumes.Then, the 1N NaOH/1MNaCl with 3 column volumes sucks with pump, and lasts 1-6 days and cultivate.If in 6 days, do not use tubing string, use the Q damping fluid C of the 1N NaOH/1M NaCl of 1 column volume, 3 column volumes, the MilliQ H of 3 column volumes so 2The 20%EtOH of O and 2.5 column volumes or 10mM NaOH wash it.Every 3-5 circulation carried out the cleaning on a large scale of a tubing string so that after 1N NaOH/1M NaCl cultivates, Q tubing string cleaning buffer (0.5%Triton X-100,0.1M acetate) with 3 column volumes upwards flows its washing, cultivated 60-80 hour, with the MilliQ H of 1.5 column volumes 20 to 70%EtOH of O, 1 column volume, the 20%EtOH washing of the 70%EtOH of 5 column volumes and 2.5 column volumes.
Add 4M NaCl to the QFF pond to reach final 0.1M concentration.Make product filter Sartobind Q100X strainer (parts #Q100X), with QFF buffer B pre-equilibration to remove intracellular toxin.Collect filtrate and mark and become the QFF PoolQ that has date and lot number.In 2 hours, handle filtrate or descend storage whole night at 4 ℃.
Make QFF PoolQ by Sartobran 0.45+0.2 μ m filtration vessel (300cm 2) (parts #5231307H500B) and collection filtrate.The QFF PoolQF that filtrate being appointed as is had date and lot number.In 2 hours, handle QFF PoolQF or descend storage whole night at 4 ℃.
Analysis in the processing comprises A 276Measurement, ELISA, LAL and irreducibility SDS-PAGE analyze.
4. tubing string 2-phenyl sepharose gel HP chromatography
Phenyl sepharose gel high performance column is to obtain from GE Healthcare.Column dimension is as follows: 100mm I.D. * 9.7cm=761ml (INdEX100/500 tubing string).Throughput is the phenyl HP 4.5-9mg gross protein of every ml, is preferably the 6-8mg gross protein (based on A 276).Flow rate is 100 ml/min (linear speeds: 76.4cm/h).The Phe buffer A is made up of 20mM TRIS, 0.4M Trisodium Citrate, and pH 7.0.The Phe buffer B is made up of 10mM TRIS, and pH 7.0, has the specific conductivity of 0.9mS/cm.
Make the AKTA detector system source of reducing phlegm and internal heat.Operation " AKTA reduce phlegm and internal heat source " program 3 times: in first time during working procedure, whole damping fluid pipelines are placed in the MilliQ water, are placed among the 1N NaOH when moving for the second time then.Last 30 minutes and finish cultivation, when moving for the third time, once more all damping fluid pipelines are placed in the MilliQ water then.With the linear speed of 30cm/h, operation " PheHP reduce phlegm and internal heat source balance " program is source and balance so that the Phe tubing string reduces phlegm and internal heat: with the MilliQ H of 2 column volumes 2The 1M NaOH/1M NaCl of O, 2 column volumes washs it, cultivates 30minutes, uses the Phe buffer A balance of 4 column volumes then.
The 1.4M Trisodium Citrate is added to the ultimate density that reaches 0.4M among the QFF poolQF.At room temperature mixture is stirred roughly 1 hour with the dissolving Trisodium Citrate, and solution is warmed up to 〉=16 ℃.QFF PoolQF+ Trisodium Citrate is loaded on the tubing string.With the Phe buffer A of 4 column volumes, the 27%Phe buffer B in A of 9-17 column volume is washed tubing string then.The length of 27%Phe buffer B washing is decided on the amount that is loaded into the gross protein on the tubing string.The protein that loads is many more, and the column volume of needed 27%Phe buffer B is few more.The 48%Phe buffer B in A with 8-10 column volume is carried out elution.Once more with 100%Phe buffer B washing tubing string.Collect 48%B elution peak and be appointed as the Phe pond that has lot number.In 2 hours, carry out next step, or under 4 ℃, the pond is stored whole night.
With the upwards mobile washing Phe of the 1M NaOH tubing string of 2 column volumes, cultivated 30 minutes, with the Phe buffer A of 3 column volumes, the H of 3 column volumes 2The 20%EtOH of O and 2.5 column volumes or 10mM NaOH washing.After 3-5 the circulation,, cultivated 30 minutes, with the Phe buffer A of 3 column volumes, the H of 3 column volumes with the upwards mobile washing Phe of the 1M NaOH tubing string of 2 column volumes 2The 70%EtOH washing of O, the 0-70%EtOH that surpasses 1 column volume, 3 column volumes, and finally be stored among 20%EtOH or the 10mM NaOH.
Analysis in the processing comprises A 276Measurement, ELISA, LAL and irreducibility SDS-PAGE analyze.
The prescription of the main body GH during 5.UF/DF II-handles
Following strainer is used for described program: Sartorius Sartocon Slice 10K Hydrosart box, 1000cm 2Other parameters comprise: the feed pressure of the filtrate of 50-90ml/min (penetrant) flow rate, 20-27psi and the retention pressure of 3-4psi.UF/DF II damping fluid is made up of 10mM sodium phosphate, 20g/L glycine and 5g/L mannitol, and pH 7.0.
, and allowed circulation 30-45 minute the system source of reducing phlegm and internal heat with 1N NaOH.Drop to below 8 up to the pH value with 4 liters MilliQ water rinse-system roughly.Last at least 5 minutes with UF/DF II damping fluid and finish balance.
The Phe pond is tapered to roughly 700-900ml (or in the retention flask, roughly 500-700ml).UF/DF II damping fluid with 4.2-5.4 liter (6-volume) is finished filter.Make retention recirculation 3-5 minute, and collect retention.With the UF/DF II damping fluid rinse-system of 100-200ml, and with washing fluid and retention combination.With Sartobran 0.45+0.2 μ m container (150cm 2) (parts #5231307H400B) filter the sample through combination, and filtrate is appointed as Y35pAF-pBx and is called " main body in the processing ".
The protein concn of Y35pAF-pBx is by measuring the A of diluted sample 276, use A 276 1mg/ml=1.037 determine.Main body in the processing can be stored down at 4 ℃ and be reached for 1 week.For carrying out prolonged storage, with its five equilibrium and remain on-80 ℃.
With MilliQ water rinse-system and by circulation 30-45 minute, with 1N NaOH cleaning.Then, with MilliQ water it is washed up to the pH value below 8.Box is stored among the 0.1N NaOH.
Analysis in the processing comprises RP-HPLC, A 276Measurement, ELISA, LAL and irreducibility SDS-PAGE analyze.
6.UF/DF the concentration of IIa-Pegylation and buffering fluid exchange
Following strainer is used for described program: Sartoiius Sartocon Slice 10K Hydrosart box, 200cm 2Other parameters comprise: the filtrate of 12-14ml/min (penetrant) flow rate, the roughly feed pressure of 25psi and the retention pressure of 0-0.5psi.Reaction buffer is made up of 20mM sodium acetate, 20g/L glycine, 5g/L mannitol, 1mMEDTA, and pH 4.0, has the specific conductivity of 2.6mS/cm.
, and allowed circulation 30-45 minute the system source of reducing phlegm and internal heat with 1N NaOH.Drop to below 8 up to the pH value with 2 liters MilliQ water rinse-system roughly.Last at least 5 minutes with reaction buffer and carry out balance.
By adding 10% acetate of 3.7% (v/v), a certain amount of pH value from the main body in the processing of step 5 is adjusted to roughly 4.Then, based on the amount of employed initial hGH, it is tapered to the target volume with 8mg/ml concentration.Reaction buffer with 5 volumes is filtered sample thoroughly.Make retention recirculation 3-5 minute, and collected retention then.Make up with the reaction buffer rinse-system of 80-120ml and with retention.Make retention filter Sartobran 0.45+0.2 μ m container (150cm through combination 2) (parts #5231307H400B).The Y35pAF-pBx/pH4 that filtrate being appointed as is had the date.Sample can be stored whole night down at 4 ℃.
The protein concn of Y35pAF-pBx/pH4 is by measuring the A through the sample of 20 times of dilutions 276, use A 276 1 Mg/ml=1.037 determine.By with the dilution of reaction buffer, with the concentration adjustment of Y35pAF-pBx/pH4 to 7mg/ml (5-9mg/ml).
7. pegylation reaction
To use the molecular weight that ethanoyl-phenylalanine is replaced the hGH of tyrosine (Y35paF) be 22 at 35 places in the position, 149Da, and the mPEG-oxyamine batch molecular weight be 30,961Da.Concerning the sequence of the ripe hGH of wild-type, referring to the SEQ ID NO:2 of No. the 2005/0170404th, U.S. Patent Publication case.Fig. 5 shows the chemical structure of employed PEG.Use the molar ratio of PEG: Y35pAF=5, calculate the amount of needed 30K MPEG-oxyamine.Slowly add 7mg/ml Y35pAF solution with the PEG powder weighing and under 25-28 ℃ to, stir simultaneously.The solid PEG artificially of bulk is smashed.After last once interpolation, under stirring gently, place 28 ℃ to last 39-50 hour reaction mixture.Be reflected at and form the oxime key between hGH and the PEG.
Analysis in the processing comprises that the irreducibility SDS-PAGE that confirms Pegylation analyzes.
8. tubing string 3-source electrode 30Q chromatography
Source electrode 30Q obtains from GE Healthcare.Column dimension is as follows: 70mm I.D. * 17.5cm=673ml (INdEX70/500 tubing string).Throughput is every ml source electrode Q 2.4mg (1-2.8mg) GH.Flow rate is 80 ml/min (linear speeds: 125cm/h).Source electrode Q buffer A is made up of 5mM TRIS, and pH 7.0.Source electrode Q buffer B is made up of 5mM TRIS, 0.1M NaCl, and pH 7.0.
For making the AKTA detector system source of reducing phlegm and internal heat, operation " AKTA reduce phlegm and internal heat source " program 3 times:, whole damping fluid pipelines are placed in the MilliQ water, and when moving for the second time, are placed among the 1N NaOH in first time during working procedure.Last 30 minutes and finish cultivation, and when moving for the third time, once more all damping fluid pipelines are placed in the MilliQ water.For making source electrode Q tubing string reduce phlegm and internal heat source and balance, operation " source electrode Q reduce phlegm and internal heat source balance " program: with the MilliQ H of 2 column volumes 2The 1M NaOH/1M NaCl washing source electrode Q tubing string of O, 2 column volumes was cultivated 30 minutes, with the source electrode Q buffer B washing of 5 column volumes, used the source electrode Q buffer A balance of 5 column volumes then.
The 0.5M TRIS alkali of 20% (v/v) is added to from the previous steps (reaction mixture of step 7).Then, make sample pass through Sartobran 0.45+0.2 μ m filtration vessel (150cm 2) (parts #5231307H400B).With the source electrode Q buffer A of 9-volume and the MilliQ H of 10-volume 2O carries out 20 times of dilutions.Then diluted sample is loaded on the tubing string.Source electrode Q buffer A washing tubing string with 5 column volumes.Linear gradient with the 0-50% source electrode Q buffer B that surpasses 10 column volumes is carried out elution.Collect cut with 1/5 column volume/cut roughly.On first main peak, carry out SE-HPLC and irreducibility SDS-PAGE analysis with cell.The cut that is compiled is appointed as the source electrode Q pond that has date and lot number.Under 4 ℃, the pond is stored whole night.
9.UF/DF (ultrafiltration/saturating filter) III-is through the concentrated and buffering fluid exchange of the main body of allotment
Use following strainer: Sartorius Sartocon Slice 10K Hydrosart box, 200cm 2Other parameters comprise: the filtrate of 12-14ml/min (penetrant) flow rate, roughly feed pressure and the retention pressure of 0-0.5psi roughly of 25psi.
, and allowed circulation 30-45 minute the system source of reducing phlegm and internal heat with 1N NaOH.Drop to below 8 up to the pH value with 2 liters MilliQ water rinse-system roughly.Then, last at least 5 minutes with the prescription damping fluid and carry out balance.
Based on the amount of employed initial substance, source electrode Q pond (step 3.6) is concentrated to target volume with 8mg/ml concentration.Carry out saturating filter with the prescription damping fluid of 6 volumes.Make retention recirculation 3-5 minute, and collect retention.Make up with the prescription damping fluid rinse-system of 50-100ml and with retention.In Biosafety ventilating kitchen or 100 grades of ventilating kitchens (Class 100hood), use Aseptic technique, with Sartobran 0.45+0.2 μ m container (150cm 2) retention of (parts #5231307H400B) sterile filtration through making up.Aseptic sample is appointed as the PEG30-pY35pAF that has lot number.
The equivalent hGH concentration of PEG30-pY35pAF is by measuring the A of diluted sample 276, by using A 276 1 Mg/ml=1.145 determine, carry out three dilutions and measurement.PEG30-pY35pAF can store down at 4 ℃ and reach 3 days.For carrying out prolonged storage, with its five equilibrium and remain on-80 ℃.
Used described scheme to handle material from the bacterial strain of W3110.Employed bacterial strain is to construct body through quadrature tRNA, quadrature aminoacyl tRNA synthetase and hGH to change shape.Analyze PEG-Y35pAF purity＞95% based on HPLC and SDS-PAGE.
Discharge the attribute that calibrating includes, but is not limited to estimate PEG30-pY35pAF fully, such as outward appearance, dissolution time, identity and purity, effectiveness, security with include, but is not limited to the calibrating of other attributes of pH value.The testing method that is used to evaluate includes, but is not limited to, the measurement of reductibility and irreducibility SDS-PAGE, SE-HPLC, RP-HPLC, IEX-HPLC, CEX-HPLC, host cell proteins matter, the measurement of surplus DNA, the A of concentration 276, cell proliferation calibrating, LAL, pyrogen, sterility, biological load (microbial limit), Karl Fisher (water-content), inclusion homogeneity and penetration degree.
The damping fluid that is used for the buffer exchange of UF/DF III can be any suitable damping fluid.Extra step behind UF/DFIII includes, but is not limited to lyophilize.Lyophilize can be used to the standard technique known to the those skilled in the art and carry out.
Carry out described method with the bacterial cell bead of about 2.7kg.
Example 7
Extra method
The purity check that is undertaken by SDS-PAGE
Following method is in order to by SDS-PAGE, then by gross protein dyeing, and the purity of main body reorganization hGH during evaluation is handled and final and PEG-reorganization hGH binding substances.In the time of in being placed on electric field, will move such as proteinic any charged molecule.The travelling speed of protein in electric field decided on net charge on strength of electric field, the protein and friction resistance.Friction resistance is the function of proteinic size and dimension.When sex change in the presence of excessive SDS, most protein with the constant weight ratio in conjunction with SDS so that it has equal electric density basically and moves in polyacrylamide gel according to the protein size.Can pass through coomassie brilliant blue staining (Coomassie Brilliant Blue staining) by the isolating protein of gel electrophoresis detects.
The equipment that is used for described program comprises following equipment and its Equivalent: XCell Surelock Mini-Cell (Invitrogen), is set to+heat block, power supply (up to 200V), little whizzer (such as little whizzer 18 of BeckmanCoulter or 22R) and the reciprocal shaker of 70-80 ℃.Reagent comprises NuPAGE MOPS SDS running buffer (20X, Invitrogen PN NP0001); NuPAGE MES SDS running buffer (20X, Invitrogen PNNP0002); NuPAGE LDS sample buffer (4X, Invitrogen PN NP0007); NuPAGE sample reductive agent (10X, Invitrogen PN NP0009); 12%Bis-Tris NuPAGE precast gel, 1.0mm * 10-hole (Invitrogen PN NP0341BOX); 4-12%Bis-Tris NuPAGE precast gel, 1.0mm * 10-hole (Invitrogen PN NP0321BOX); Pre-staining molecular weight marker thing (SeeBlue Plus2, Invitrogen PNLC5925); MilliQ quality H 2O (MilliQ-quality H 2O) or Equivalent; SimplyBlue SafeStain (Invitrogen PN LC6065) or Equivalent; Reference standard (WHO rhGH standard); The calibration solution of rhGH (Y35pAF-pB2/pB3,2mg/ml); The calibration solution of pEG-rhGH binding substances (PEG30-pY35pAF-01,2mg/mL).The protein concn of standard and test article is to use known standard technique measurement in affiliated field.
The analysis of pre-polyoxyethylene glycol purification step sample
Preparation 3 μ g reference standards (RS for example calibrates solution Y35pAF-pB2/pB3) under the irreducibility condition.Add 3 μ g reference standards to 4X LDS and MilliQ H 2Among the O to obtain 28 μ l samples at 1X LDS.Similarly, preparation rhGH test article under the irreducibility condition.Under+70-80 ℃, rhGH tested the heating of article and reference standard 8-10 minute and centrifugally be loaded on the gel then.According to the explanation of manufacturers, prepare 12%Bis-Tris NuPAGE precast gel with 1X MOPS SDS running buffer.Following loading gel: pre-staining molecular weight marker thing, 3 μ g reference standards, test article, and under the maximum of 200V is set, move 50 minutes.In deionized water, cultivate gel, shaking use SimplyBlue or Equivalent dyeing down, and using water decolorization.RhGH is tested the master tape position of article and the master tape position comparison of 3 μ g reference standards.
The analysis of main body rhGH in the purified processing
The reference standard (RS, for example WHO rhGH) of preparation 20 μ g and 1 μ g under irreducibility and reductive condition.Concerning the irreducibility condition, add 20 or 1 μ g reference standard to 4X LDS and MilliQ H 2Among the O in 1X LDS, to obtain 28 μ l samples.Concerning reductive condition, add 20 or 1 μ g reference standard to 4X LDS, 10X reductive agent and MilliQ H 2Among the O in 1X LDS and 1X reductive agent, to obtain 28 μ l samples.Under+70-80 ℃, rhGH was tested article and reference standard heating 8-10 minute and centrifugal before being loaded on the gel.According to the explanation of manufacturers, in 1X MOPS SDS running buffer, a unit that is used for the irreducibility condition moves 12%Bis-Tris NuPAGE precast gel with another unit that is used for reductive condition.Following loading gel: pre-staining molecular weight marker thing, 1 μ g reference standard, 20 μ g reference standards, blank swimming lane, then test article, under the maximum setting of 200V, last 50 minutes.In deionized water, cultivate gel, shaking use SimplyBlue or Equivalent dyeing down, and using water decolorization.RhGH is tested the master tape position of article and the master tape position comparison of 20 μ g reference standards.In the swimming lane of rhGH test article, except that master tape, do not have than the intensive band more of the master tape in the swimming lane of 1 μ g reference standard (5%).
The analysis of the Pegylation of rhGH and the purifying of PEG-rhGH
Under the irreducibility condition, prepare reference standard (RS for example calibrates solution PEG30-pY35pAF-01).Add the PEG30-pY35pAF-01 of 5 μ g to 4X LDS and MilliQ H 2Among the O in 1X LDS, to obtain final 28 μ l samples.Decide on the program of being analyzed, add the test article of 5-20 μ g to 4X LDS and MilliQ H 2Among the O in 1X LDS, to obtain final 28 μ l samples.Concerning the pegylation reaction mixture, use the test article of 15-20 μ g.Concerning the analysis of pegylation reaction mixture: a) add PEG allowing before estimation is retained in the relative percentage without the rhGH of Pegylation in the pegylation reaction mixture, continuously the rhGH of concentration pH 4 times; B) compare between 1/10 diluent of the reaction mixture of 10 μ L.During purifying behind the Pegylation, use test article from the 5-20 μ g of tubing string cut.Concerning the analysis of PEG-rhGH tubing string cut, the tubing string cut is to come relatively by each tubing string cut (each tubing string cuts of common 21 μ L) that uses fixed volume.
Do not heat PEG-rhGH test article or PEG-rhGH reference standard sample.Sample is centrifugal and be loaded into explanation according to manufacturers, on the 4-12%Bis-Tris NuPAGE precast gel with the preparation of 1X MES SDS running buffer.Following loading gel: pre-staining molecular weight marker thing, 5 μ g reference standards, then test article and set operation 35 minutes with the maximum of 200V.In deionized water, cultivate gel, shaking use SimplyBlue or Equivalent dyeing down, and using water decolorization.
The electrophoretogram of PEG-rhGH test article will meet the electrophoretogram that obtains with the PEG-rhGH reference standard.
Final analysis through the rhGH of Pegylation product
The reference standard (RS for example calibrates solution PEG30-pY35pAF-01) of preparation 10 μ g under irreducibility and reductive condition.Add the PEG30-pY35pAF-01 (2mg/mL) of 10ug to 4X LDS and MilliQ H 2Among the O in 1X LDS, to obtain final 28 μ l samples.Concerning reductive condition, add 10 μ g reference standards to 4X LDS, 10X reductive agent and MilliQ H 2Among the O in 1X LDS and 1X reductive agent, to obtain 28 μ l samples.Similarly, the rhGH through Pegylation that also prepares 10 μ g under irreducibility and reductive condition tests article.Do not heat PEG-rhGH test article and PEG-rhGH reference standard, but in the explanation that is loaded into according to manufacturers, before on the 4-12%Bis-Tris NuPAGE precast gel of 1X MES SDS running buffer preparation, that it is centrifugal rapidly.With pre-staining molecular weight marker thing, 10 μ g reference standards, blank swimming lane (recommending in order to minimize the possible effect of carrying), the order of then testing article loads gel, lasts 35 minutes with the maximum set value of 200V simultaneously.In deionized water, cultivate gel, shaking use SimplyBlue or Equivalent dyeing down, and using water decolorization.
The electrophoretogram of PEG-rhGH test article should meet the electrophoretogram that obtains with the PEG-rhGH reference standard.The electrophoretogram of PEG-rhGH test article should meet the electrophoretogram that obtains with the PEG-rhGH reference standard.Any band of the reference standard that do not match may be degraded product or aggregation.Higher molecular weight band can be represented aggregation, and lower molecular weight band can be represented no longer and PEG bonded polypeptide.
Purity and the chemical degradation analysis of the rhGH that is undertaken by CEX-HPLC/IEX-HPLC
Following method is in order to by cationic exchange high performance liquid chromatography (CEX-HPLC), and evaluation is through the relative purity of the recombinant human growth hormone (rhGH) of Pegylation and possible chemical degradation (that is to say deamidating).CEX-HPLC is the technology that relies on protein and be fixed in the electric charge-coulombic interaction between the electric charge on the resin.The ion of the proteinic positively charged of cation-exchange chromatography utilization and electronegative resin-bonded.The ordinary construction of the rhGH deamidating of asparagine (Asn) residue changes and described CEX-HPLC method, permission through Pegylation with without the rhGH of Pegylation through the deamidating thing with the separating of deamidating intermediate.Described method is in order to support identification and the purity assessment through the rhGH of Pegylation.Use described technology to can be observed the some parts degraded product of rhGH.
The equipment that is used for described program comprises following equipment and its Equivalent: UV/Vis spectrophotometer (Agilent 8453 or Equivalent); 50 μ l quartz colorimetric utensils; 0.5mL the Vivaspin thickener (if desired; Vivascience 10,000MWCO, PES, VS0102 or Equivalent); PD-10, NAP-10 or NAP-5 tubing string (GE Healthcare, catalog number (Cat.No.) #17-0851-01,17-0853-01,17-0854-01); HPLC bottle and lid (Alltech 100 μ l nut polypropylene bottle #12962, TFE screen casing lid #73048, perforate nut #73044 or Equivalent); Clean 1L and 2L vial; Tubing string-PolyCAT A 4.6 * 200mm, 5 μ, 1000
Figure A20058004446301471
(PolyLC, 204CT0510) with PolyCAT A guard column, 4.6 * 10mm, 5 μ, 1000 (PolyLC, JGCCT0510); Can carry out the high pressure liquid chromatography utensil (such as Agilent 1100 HPLC that are equipped with vacuum degasser, level Four gradient pump, constant temperature self-actuated sampler, constant temperature column compartment, diode-array detector (DAD) and Chemstation chromatogram software) of linear gradient.
Unless otherwise indicated, otherwise the reagent that is used for described program comprise water (Milli-Q quality or Equivalent) and solid state chemistry product be AG or better and solvent be HPLC level or better.Unless otherwise instructed, otherwise the storage of reagent and programstep are at room temperature to take place.The example of described chemical comprises ammonium acetate, Spectrum A2149, HPLC level or Equivalent; Acetonitrile, Fisher A998, HPLC level or Equivalent; Bicarbonate of ammonia, Fluka # 09830, Ultra＞99.5% or Equivalent; Glacial acetic acid, Fisher#64-19-7, HPLC level or Equivalent; Two hydration Trisodium Citrates, SpectrumS0165, USP level or Equivalent; Glycine, Spectrum AM125 or Equivalent; Mannitol, Spectrum MA165 or Equivalent; 6N HCl, Mallinckrodt 2662-46 or Equivalent.
Mobile phase A damping fluid is that 50mM ammonium acetate, 40% acetonitrile (AcCN) and the mobile phase B damping fluid of pH 4.25 is 500mM ammonium acetate, the 40%AcCN of pH 4.25.Prepared extra reagent is 10% acetate; The 30mM bicarbonate of ammonia of the damping fluid that is used for deamidating: pH 9.0; With the diluted sample damping fluid: the 20mM Trisodium Citrate, the 20g/L glycine, the 5g/L mannitol, pH 6.0, use 0.22 μ m PES strainer (Corning#431098 or Equivalent) with each reagent sterile filtration.
With the rhGH of The World Health Organization (WHO) (catalog number (Cat.No.) #98/574) as without the hGH standard of Pegylation.In the water of 1.0ml, its rehydration and use dilution buffer liquid are diluted to 1.1mg/ml.10% acetate that adds 10% (v/v) is to obtain pH value between pH 3.8-4.3 and the ultimate density (acceptable scope is 0.9-1.1mg/ml) of 1.0mg/ml.Prepare another hGH standard in a similar manner, promptly calibrate solution Y35pAF-pB2/pB3 without Pegylation.Also prepare standard in a similar manner, promptly calibrate solution PEG30-pY35pAF-01 through the hGH of Pegylation.
Concerning through the separation solution (Resolution Solution) of Pegylation, use PD-10, Nap-10 or Nap-5 desalination tubing string that PEG30-pY35pAF-01 is calibrated the solution buffering and be replaced with 30mM bicarbonate of ammonia, pH 9.0 damping fluids.Use 0.5mL Vivaspin thickener that standard is concentrated to roughly 2mg/ml (acceptable scope is 1.9-2.1mg/ml), and under 37 ℃ with sample cultivation 24 hours.Use dilution buffer liquid that the part of required sample or sample is diluted to 1.1mg/ml, and 10% acetate that adds 10% (v/v) is to obtain pH value between pH 3.8-4.3 and the ultimate density (acceptable scope is 0.9-1.1mg/ml) of 1.0mg/ml.
Use dilution buffer liquid will test article and be diluted to 1.1mg/ml, and 10% acetate that adds 10% (v/v) is to obtain pH value between pH 3.8-4.3 and the ultimate density (acceptable scope is 0.9-1.0mg/ml) of 1.0mg/ml.Use is known standard technique measurement standard and the protein concn of testing article in affiliated field.
Program
Set up utensil: 1) tubing string: PolyCAT A 204CT0510 and JGCCT0510 with following condition; 2) self-actuated sampler temperature: room temperature; 3) pump is set: the 81.5-108.5mM ammonium acetate (7-13%B) of stepping gradient: pH 4.25, then the 108.5-500mM ammonium acetate (13-100%B) of pH 4.25; 4) table 4;
5) syringe is set--injection: standard injection; Volume injected: 25 μ l; Pumping velocity: 50 μ l/min; Injection speed: 50 μ l/min; Pin washing: 15 μ l H 2O; Stand-by time: the same with pump; 6) DAD signal: table 5;
Figure A20058004446301482
Peak width:＞0.1min; Slit: 4nm; Stand-by time: the same with pump; 7) tubing string thermostatted: temperature: 30 ℃; The record temperature.
100% mobile phase A with 10-15 column volume makes the tubing string balance.The calibration solution PEG30-pY35pAF-01 of injection 25-50 μ l through Pegylation.56.97min (during ± 0.5min) retention time, the peak that elution is main through Pegylation.Then, the WHO of injection 25-50 μ l or calibration solution Y35pAF-pB2/pB3 and operation HPLC program.98.54min (± 0.5min) retention time, promptly the time concerning main 1.73 ± 0.01 relative retention time through the peak of Pegylation, the peak that elution is main without Pegylation.
Then, the separation solution of injection 25-50 μ l through Pegylation.In the color atlas that is obtained, 56.97min (during ± 0.5min) retention time, elution main through the peak of Pegylation, and with respect to main peak (45.23 ± 0.3min; During 0.79 ± 0.02 the retention time of (current condition causes 2.3 ± 0.02 resolution), elution is through the desamido-base peak of Pegylation.
Inject the test article of 25-50 μ l then through Pegylation, and operation HPLC program.With sample operation three times and write down average retention time.Color atlas is to produce with absorbancy (280nm).
Data analysis
To compare with calibration solution PEG30-pY35pAF-01 through the retention time of the rhGH of Pegylation test article.Use: (integral areas at the integral area of main peak/all peaks) * 100% calculates the average purity of test article.Ignore any peak that produces owing to solvent.
The purity testing of the rhGH that is undertaken by SEC-HPLC
Described program is in order to by size exclusion high performance liquid chromatography (SEC-HPLC), assessment comprise in the processing material and through the purity of the recombinant human growth hormone (rhGH) of the rhGH of Pegylation.Described test with monomer from sample, and through the sample of Pegylation with separate with other related substances without the dimer in the sample of Pegylation with higher molecular weight.SEC-HPLC is to use the technology of stationary phase as the porous matrix of mobile phase molecule infiltration.Enough for a short time, be delayed with the sample molecule that enters vesicular structure, bigger molecule is excluded and therefore is transported apace and passes through tubing string simultaneously.Therefore, the size exclusion chromatography, meaning be by apart molecule and chromatogram elution time be the feature of concrete molecule.Described program is in order to measure the per-cent of monomer (through Pegylation with without Pegylation) rhGH.Use described technology to can be observed dimer and other high molecular weight proteins.
The reference of described technology comprises 2002, the 193 pages of European Pharmacopoeia; 2001, the 1941 pages of BritishPharmacopoeia; " High-Performance Size-ExclusionChromatographic Determination of the Potency of Biosynthetic Human Growth HormoneProducts " .Journal of Chromatography 435 (1988) of people such as R.M.Riggin, the 307-318 page or leaf.
The equipment that is used for described program comprises following equipment and its Equivalent: UV/Vis spectrophotometer (Agilent 8453 or Equivalent); 50 μ l quartz colorimetric utensils; 0.5mL the Vivaspin thickener (if desired; Vivascience 10,000MWCO, PES, VS0102 or Equivalent); HPLC bottle and lid (Alltech 100 μ l nut polypropylene bottle #12962, TFE screen casing lid #73048, perforate nut #73044 or Equivalent); Clean 1L and 2L vial; Tubing string-Tosohaas TSK Super SW3000 18675 and Super SW guard column 18762, has the size of 4.6 * 300mm, the granularity of 4 μ m and 250
Figure A20058004446301491
The aperture based on the size exclusion HPLC tubing string of silica guard column together with size with 4.6 * 35mm and 4 μ granularities; Can carry out the high pressure liquid chromatography utensil (such as the Agilent 1100HPLC that is equipped with vacuum degasser, level Four gradient pump, constant temperature self-actuated sampler, constant temperature column compartment, diode-array detector (DAD), refractive index detector (RID) and Chemstation chromatogram software) of linear gradient.
Unless otherwise indicated, otherwise the reagent that is used for described program comprise water (Milli-Q quality or Equivalent) and solid state chemistry product be AG or better and solvent be HPLC level or better.Unless otherwise instructed, otherwise the storage of reagent and programstep are at room temperature to take place.The example of described chemical comprises a hypophosphite monohydrate sodium dihydrogen, SpectrumU.S.P. level S0130 or Equivalent; Seven hypophosphite monohydrate disodium hydrogens, Spectrum U.S.P. level S0140 or Equivalent; The 2-propyl alcohol, Fisher HPLC level A451-4 or Equivalent.
The mobile phase damping fluid is the 63mM sodium phosphate of 97% pH 7.0,3% 2-propyl alcohol.Solution A is the 25mM sodium phosphate of pH 7.0.Use 0.22 μ m PES strainer (Corning#431098 or Equivalent) with two kinds of solution sterile filtrations.
The rhGH of The World Health Organization (WHO) (catalog number (Cat.No.) #98/574) is as the hGH standard without Pegylation.Water with 1.0ml is diluted to 1mg/ml concentration (acceptable scope is 0.9-1.1mg/ml) with its rehydration and in the WHO damping fluid.Prepare another hGH standard in a similar manner, promptly calibrate solution Y35pAF-pB2/pB3, and with 20mM Trisodium Citrate, 2% glycine, 0.5% mannitol, pH 6 dilutes without Pegylation.Also prepare standard in a similar manner, promptly calibrate solution PEG30-pY35pAF-01, and with 20mM Trisodium Citrate, 2% glycine, 0.5% mannitol, pH 6 dilutes through the hGH of Pegylation.Concerning separation solution, make PEG30-pY35pAF-02 higher molecular weight standard reach 1mg/ml concentration (acceptable scope is 0.9-1.1mg/ml).Described solution contains roughly 33%PEG-PEG-GH, 66.5%PEG-GH.With solution A test substances is diluted to roughly 1.0mg/ml (acceptable scope is 0.9-1.1mg/ml).Known standard technique is measured all sample concentrations in the affiliated field of use.Can carry out dilution of sample with any suitable damping fluid.
Program
Set up utensil: 1) tubing string: TSK Super SW300018675 and guard column 18762 with following condition; 2) pump is set--gradient: isoconcentration; Flow rate: 0.3ml/min; Time length: 25min; Peak pressure: 120 crust; 3) syringe is set--injection: standard injection; Volume injected: 10 μ l; Pumping velocity: 100 μ l/min; Injection speed: 100 μ l/min; Pin washing: 100 μ l H 2O; Stand-by time: the same with pump; 4) DAD signal: table 6;
Figure A20058004446301501
Peak width:＞0.05min; Slit: 2nm; Stand-by time: the same with pump; 5) RID signal--temperature: 35 ℃; Time of response:＞0.2min 4s, standard; 6) tubing string thermostatted: temperature: 23 ℃; The record temperature.
With 10 column volumes (under 0.3ml/min, mobile mutual-assistance tubing string balance 50ml=166min), and before injected sample, purify RID at least 20 minutes.Before the sample operation, make DAD and RI detector self-poise.
Inject the calibration solution Y35pAF-pB2/pB3 (or WHO standard) of 20 μ l, and operation HPLC program.In the color atlas that is obtained, when the retention time of 12.96 (± 0.05) min roughly, the peak that elution is main without Pegylation.With respect to 0.94 ± 0.02 retention time of main peak the time, the rhGH dimer without Pegylation of elution higher molecular weight.When the retention time of 7.3-8.0min, the aggregation of elution higher molecular weight.
Inject the calibration solution PEG30-pY35pAF-01 of 20 μ l.When the retention time of 8.33 (± 0.08) min roughly (concerning 0.64 relative retention time without the rhGH of Pegylation), the peak that elution is main through Pegylation.In time greater than 8.0min, the elution higher molecular weight through the rhGH of Pegylation aggregation.
Inject the separation solution of 20 μ l, and operation HPLC program.When the retention time of 8.28min elution main through the peak of Pegylation, and at 7.54min, during promptly with respect to the relative retention time of 0.9 (± 0.05) at main peak through Pegylation, the material of elution higher molecular weight.
Inject the test article of 20 μ l, and operation HPLC program.With sample operation three times and write down average retention time.RhGH being tested the retention time of article compares with the rhGH standard.
The SEC-HPLC data of the article of self-test are in the future compared with the data that obtain from reference standard.For measuring the purity without the rhGH of Pegylation, rhGH is tested the integration main peak area of article and compare with total peak area, and pass through: (the main peak area/total peak area of rhGH sample) * 100% calculates the monomeric per-cent in the rhGH test article.The dimer in the calculating hGH test article and/or the per-cent of higher aggregation.Ignore because any peak that solvent exists.For measuring purity through the rhGH of Pegylation, to compare with total peak area through the integration main peak area of the rhGH of Pegylation sample, and pass through: (the main peak area/total peak area of PEG-rhGH sample) * 100% calculates the monomeric per-cent through Pegylation in the PEG-rhGH sample.The dimer through Pegylation of calculating in the hGH of Pegylation test article, higher aggregation and without the monomeric per-cent of Pegylation.Ignore because any peak that solvent exists.Before the hGH peak of Pegylation, the material of higher molecular weight is represented at the peak of elution in color atlas main.The material of described higher molecular weight can include, but is not limited to dimer (such as PEG-PEG-hGH and other possible dimers) or solubility is assembled thing.The material of representing lower molecular weight at the peak of main elution after the hGH peak of Pegylation.The material of described lower molecular weight can include, but is not limited to the hGH through Pegylation without the monomer of Pegylation and clipped form.
Purity and the chemical degradation analysis of the rhGH that is undertaken by RP-HPLC
Following method is in order to by C4 anti-phase high performance liquid chromatography (RP-HPLC), assesses the relative purity of recombinant human growth hormone (rhGH) and possible chemical degradation (deamidating and oxidation).RP-HPLC is the technology according to relative hydrophobicity isolated molecule.Sample is delivered to covalently on the stationary phase with the silica of hydrocarbon chain bonding.Postpone the molecule paid close attention to and with the elution of isoconcentration solvent by stationary phase.The chromatogram elution time is the characteristic of concrete molecule.Described method based on hydrophobicity with separate rhGH with nuance such as the relevant retention behavior aspect of the structural modification of deamidating.Described method is in order to the identification of supporting rhGH and purity assessment.Use described technology to can be observed the some parts degraded product of rhGH.
The reference of described technology comprises 2002, the 193 pages of European Pharmacopoeia; BritishPharmacopoeia 2001, the 1938-1939 pages or leaves; People's such as R.M.Riggin A Reversed-Phase HighPerformance Liquid Chromatographic Method for Characterization of Biosynthetic HumanGrowth Hormone " Analytical Biochemistry 167,199-209 (1987).
The equipment that is used for described program comprises following equipment and its Equivalent: UV/Vis spectrophotometer (Agilent 8453 or Equivalent); 50 μ l quartz colorimetric utensils; PD-10, Nap-10 or Nap 5 (decide on sample volume; GE HealthcareNap5 tubing string 17-0853-02 or Equivalent); 0.5mL the Vivaspin thickener (if desired; Vivascience 10,000MWCO, PES, VS0102 or Equivalent); HPLC bottle and lid (Alltech 100 μ l nut polypropylene bottle #12962, TFE screen casing lid #73048, perforate nut #73044 or Equivalent); Clean 1L and 2L vial; Tubing string-Vydac C4 214ATP54 has the size of 4.6 * 250mm, the granularity and 300 of 5 μ
Figure A20058004446301521
The C4-silica anti-phase HPLC tubing string in aperture; Can carry out the high pressure liquid chromatography utensil (such as Agilent 1100 HPLC that are equipped with vacuum degasser, level Four gradient pump, constant temperature self-actuated sampler, constant temperature column compartment, diode-array detector (DAD) and Chemstation chromatogram software) of linear gradient.
Unless otherwise indicated, otherwise the reagent that is used for described program comprise water (Milli-Q quality or Equivalent) and solid state chemistry product be AG or better and solvent be HPLC level or better.Unless otherwise instructed, otherwise the storage of reagent and programstep are at room temperature to take place.The example of described chemical comprises the TRIS-trometamol, U.S.P. level, Spectrum TR149 or Equivalent; The N-propyl alcohol, HPLC level, 99.9%, Sigma Aldrich 34871 or Equivalent; Bicarbonate of ammonia, Ultra＞99.5%, Fluka#09830 or Equivalent.
The damping fluid that is used for deamidation control is the 30mM bicarbonate of ammonia of pH 9.0.The damping fluid that is used for oxidation control is the 50mM TRIS of pH 7.5.Use 0.22 μ m PES strainer (Corning # 431098 or Equivalent) with each the solution sterile filtration in the described solution.Mobile phase: 710ml 50mM TRIS-HCl pH 7.5; 290ml n-propyl alcohol (or having other proper volume of 50mM Tris-HCl and 29% n-propyl alcohol of 71%pH 7.5).6.05g trometamol (USP level, Spectrum # TR149 or Equivalent) is dissolved in 0.95L Milli-Q H 2Among the O.Make solution reach pH 7.5 and use Milli-Q H with HCl 2O makes volume reach 1L.After 2 kinds of solvents (TRIS and propyl alcohol) mixing, use 0.22 μ m PES strainer (Corning # 431098 or Equivalent) with mixture sterile filtration.Regulator solution is 50%AcCN:H 2O, 0.1%TFA.
Sample as standard comprises that the water rehydration through 1.0ml arrives the rhGH of The World Health Organization (WHO) (catalog number (Cat.No.) #98/574) of 1.9-2.1mg/ml and the rhGH reference standard of 1.9-2.1mg/ml concentration.The deamidation separation solution is by using PD-10, Nap-10 or Nap-5 desalination tubing string (decide on sample volume), WHO standard is cushioned be replaced with the 30mM ammonium bicarbonate buffers of pH 9.0 and make.Use 0.5mL Vivaspin thickener that standard is concentrated to 1.9-2.1mg/ml, and under 37 ℃ with sample cultivation 24 hours.Concerning the oxidation separation solution, use PD-10, Nap-10 or Nap-5 desalination tubing string (deciding) on sample volume, the WHO standard buffering is replaced with the 50mMTRIS damping fluid of pH 7.5.Use 0.5mL Vivaspin thickener that standard is concentrated to 1.9-2.1mg/ml and adds H 2O 2Reach 0.015% ultimate density.Under 4 ℃, will react and cultivate 24hrs.By adding 0.5-1 μ l (if 20mg/ml) catalase reaction is stopped.Be specimen, test substances is diluted to the 2.0mg/ml protein concn.
Program
Set up the following condition utensil that has: (1) tubing string: Vydac C4214ATP54 tubing string; 2) pump is set--gradient: isoconcentration; Flow rate: 0.5ml/min; Time length: 60min; Peak pressure: 200 crust; 3) self-actuated sampler temperature: 4 ℃; 4) syringe is set--injection: standard injection; Volume injected: 20 μ l; Pumping velocity: 100 μ l/min; Pin washing: water 100 μ l; Injection speed: 100 μ l/min; Stand-by time: the same with pump; 5) DAD signal (table 7):
Figure A20058004446301531
Peak width:＞0.1min; Slit: 4nm; Stand-by time: 60min; 6) tubing string thermostatted: temperature: 45 ℃; The record temperature; 7) preliminary integration incident (Chemstation software, Agilent): slope sensitivity: 0.1; Peak width: 0.5; Area amputation (Area Reject): 1.0; Peak height amputation: 1.0; Integration when 10min.
Regulator solution (50%AcCN, H with 300mL 2O, 0.1%TFA), with between 0.5 and 1.5ml/min between flow rate with tubing string preconditioning.Should before using tubing string, carry out pre-equilibration, if or the peak broaden, use regulator solution (200-300mL) with the tubing string re-adjustment so.With 10 column volumes (under 0.5ml/min, mobile mutual-assistance tubing string balance 41.5ml=83min).
The standard of using self-actuated sampler to inject 20 μ l, and operation HPLC program.Form if the retention time of WHO standard not between 32.5-35min, is adjusted mobile phase so, make the tubing string reequilibrate and the standard of reruning.The adjustment of suggestion comprises, if retention time less than 32.5min, so every liter of mobile phase is added the 50mM Tris-HCl less than the pH 7.5 of 5ml, and if retention time greater than 35, add n-propyl alcohol so less than 2ml.Because the evaporation of propyl alcohol may take place, so move standard and therefore adjust damping fluid every day to be tested at sample.
Inject the deamidation separation solution of 20 μ l, and operation HPLC program.With respect to about 0.88 ± 0.03 retention time of main peaks the time, desamido--hGH shows as small peak.Be 0.8 to 1.8 (current condition causes 1.26 ± 0.06 resolution) corresponding to the peak-to-peak resolution of hGH and desamido--hGH for the symmetrical factor at least 1.0 (current condition causes 1.29 ± 0.04 resolution) and hGH peak.
Inject the oxidation separation solution of 20 μ l, and operation HPLC program.With respect to about 0.8 the retention time at main peak the time, oxidation-hGH shows as small peak.
Inject the test article of 20 μ l, and operation HPLC program.With sample operation three times.Write down average retention time.
Data analysis
The average retention time of test article is compared with rhGH reference standard or WHO standard.Calculate the average purity of test article: (integral areas at the integral area of main peak/all peaks) * 100%.Ignore because any peak that solvent exists.Color atlas is showed absorbancy (220nm).
Should be appreciated that, example of Miao Shuing and embodiment only are used for illustrative purpose herein, and will be, and be included in the category of the purport of the application's case and scope and accessory claim to those skilled in the art hint according to its various tangible modification or change of making.The open case of all that quote, patent and patent application case are all to incorporate this paper by reference into to be used for all purposes at this herein.
Table 8: the sequence of being quoted.
? SEQ? ID# The sequence title
?1 The full length amino acid sequence of hGH
?2 The mature amino acid sequence of hGH (with merit heterogeneous 1)
?3 The 20-kDa hGH variant of the residue 32-46 of hGH disappearance wherein
Sequence table
<110>Buechler，Ying
Lieu，Ricky
Ong，Michael
Bussell，Stuart
Knudsen，Nick
Cho，Ho?S.
＜120〉method of expression and purification of recombinant human growth hormone
<130>AMBX-0078.00PCT
<150>60/638,616
<151>2004-12-22
<150>60/655,744
<151>2005-02-23
<150>60/680,977
<151>2005-05-13
<150>60/727,968
<151>2005-10-17
<160>3
<170>PatentIn?version?3.3
<210>1
<211>217
<212>PRT
＜213〉homo sapiens
<400>1
Met?Ala?Thr?Gly?Ser?Arg?Thr?Ser?Leu?Leu?Leu?Ala?Phe?Gly?Leu?Leu
1 5 10 15
Cys?Leu?Pro?Trp?Leu?Gln?Glu?Gly?Ser?Ala?Phe?Pro?Thr?Ile?Pro?Leu
20 25 30
Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln
35 40 45
Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Pro?Lys
50 55 60
Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe
65 70 75 80
Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys
85 90 95
Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp
100 105 110
Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val
115 120 125
Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu
130 135 140
Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg
145 150 155 160
Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser
165 170 175
His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe
180 185 190
Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys
195 200 205
Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
210 215
<210>2
<211>191
<212>PRT
＜213〉homo sapiens
<400>2
Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg
1 5 10 15
Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu
20 25 30
Glu?Ala?Tyr?Ile?Pro?Lys?Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro
35 40 45
Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg
50 55 60
Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu
65 70 75 80
Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val
85 90 95
Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp
100 105 110
Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu
115 120 125
Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser
130 135 140
Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr
145 150 155 160
Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe
165 170 175
Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
180 185 190
<210>3
<211>176
<212>PRT
＜213〉homo sapiens
<400>3
Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg
1 5 10 15
Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Asn
20 25 30
Pro?Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn
35 40 45
Arg?Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser
50 55 60
Leu?Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser
65 70 75 80
Val?Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr
85 90 95
Asp?Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg
100 105 110
Leu?Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr
115 120 125
Ser?Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn
130 135 140
Tyr?Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr
145 150 155 160
Phe?Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe
165 170 175

Claims (12)

1. method, it comprises:
A) in containing non-naturally encoded amino acid whose liquid nutrient media, under the condition that helps growing,
Cultivation can be produced the recombinant host cell that comprises described non-naturally encoded amino acid whose hGH;
B) comprise the production of described non-naturally encoded amino acid whose hGH by described cell induction; With
C) comprise non-naturally encoded amino acid whose hGH from described cell or substratum purifying are described.
2. method according to claim 1, wherein one or more non-naturally encoded amino acid whose described interpolations occur in the time place that is selected from the group that is made up of the following time: continuously at the cell growing period, time before inducing is in inducing or the time after inducing.
3. method according to claim 2, wherein one or more non-naturally encoded amino acid whose described interpolations take place with inducing simultaneously.
4. method according to claim 2, wherein one or more non-naturally encoded amino acid whose described interpolations occur in and induce roughly 1 hour before.
5. method, it comprises following steps:
(a) make non-naturally encoded amino acid whose hGH and the anion exchange chromatography matrix of comprising, contact under described hGH and the described matrix bonded condition allowing from the step c) of claim 1;
(b) from described matrix elution and the described hGH of collection;
(c) make described hGH and hydrophobic interaction chromatogram (HIC) matrix, contact under described hGH and the described matrix bonded condition allowing from step b); With
(d) comprise non-naturally encoded amino acid whose pure substantially hGH from elution of described HIC matrix and the described hGH of collection to provide.
6. method according to claim 1, wherein said recombinant host cell is selected from the group that is made up of prokaryotic cell prokaryocyte and eukaryotic cell.
7. method according to claim 1, the hGH of wherein said purifying substantially is selected from the group that is made up of ripe hGH, ripe hGH variant, hGH polypeptide and hGH polypeptide variants.
8. method according to claim 5, wherein said method comprises, after step a), make described hGH and hydroxyapatite chromatography matrix from described anion-exchange chromatography elution, contact under described hGH and the described matrix bonded condition allowing, then from the additional step of elution of described hydroxyapatite chromatography matrix and the described hGH of collection.
9. method, it comprises following steps:
(a) make the polyoxyethylene glycol (PEG) that comprises non-naturally encoded amino acid whose hGH and derivatize, react being enough to form under the condition of hGH-PEG binding substances; With
(b) by making described hGH-PEG binding substances and anionresin matrix, contact under described hGH and the described matrix bonded condition allowing, then from described anion exchange chromatography matrix elution and the described hGH of collection, the hGH-PEG binding substances of purifying separates and the described hGH-PEG binding substances of purifying to provide substantially.
10. one kind is separated the method for the hGH-PEG binding substances of purifying substantially, and it comprises following steps:
A) in containing non-naturally encoded amino acid whose liquid nutrient media, under the condition that helps growing, cultivation can be produced the recombinant host cell that comprises described non-naturally encoded amino acid whose hGH;
B) comprise the production of described non-naturally encoded amino acid whose hGH by described cell induction; With
C) make from comprising of step b) non-naturally encoded amino acid whose hGH and anion exchange chromatography matrix, contact under described hGH and the described matrix bonded condition allowing, then from described matrix elution and the described hGH of collection;
D) make from the described hGH and the hydroxyapatite chromatography matrix of the described anion-exchange chromatography elution of step c), contact under described hGH and the described matrix bonded condition allowing, then from elution of described hydroxyapatite chromatography matrix and the described hGH of collection;
E) make described hGH and hydrophobic interaction chromatogram (HIC) matrix from step d), contact under described hGH and the described matrix bonded condition allowing, then, comprise non-naturally encoded amino acid whose hGH to provide from elution of described HIC matrix and the described hGH of collection;
F) make from comprising of the step e) non-naturally encoded amino acid whose hGH and the polyoxyethylene glycol (PEG) of derivatize, react being enough to form under the condition of hGH-PEG binding substances; With
G) by making described hGH-PEG binding substances and anionresin matrix, contact under described hGH and the described matrix bonded condition allowing, then from described anion exchange chromatography matrix elution and the described hGH of collection, the hGH-PEG binding substances of purifying separates and the described hGH-PEG binding substances of purifying to provide substantially.
11. one kind is separated the method for the hGH-PEG binding substances of purifying substantially, it comprises following steps:
A) in containing non-naturally encoded amino acid whose liquid nutrient media, under the condition that helps growing, cultivation can be produced the recombinant host cell that comprises described non-naturally encoded amino acid whose hGH;
B) comprise the production of described non-naturally encoded amino acid whose hGH by described cell induction; With
C) make from comprising of step b) non-naturally encoded amino acid whose hGH and anion exchange chromatography matrix, contact under described hGH and the described matrix bonded condition allowing, then from described matrix elution and the described hGH of collection;
D) make described hGH and hydrophobic interaction chromatogram (HIC) matrix from step d), contact under described hGH and the described matrix bonded condition allowing, then, comprise non-naturally encoded amino acid whose hGH to provide from elution of described HIC matrix and the described hGH of collection;
E) make from comprising of the step d) non-naturally encoded amino acid whose hGH and the polyoxyethylene glycol (PEG) of derivatize, react being enough to form under the condition of hGH-PEG binding substances; With
F) by making described hGH-PEG binding substances and anionresin matrix, contact under described hGH and the described matrix bonded condition allowing, then from described anion exchange chromatography matrix elution and the described hGH of collection, the hGH-PEG binding substances of purifying separates and the described hGH-PEG binding substances of purifying to provide substantially.
12. a method, it comprises:
A) in containing non-naturally encoded amino acid whose liquid nutrient media, under the condition that helps growing, cultivation can be produced the recombinant host cell that comprises described non-naturally encoded amino acid whose hGH;
B) comprise the production of described non-naturally encoded amino acid whose hGH by described cell induction; With
C) make from comprising of the step b) non-naturally encoded amino acid whose hGH and the polyoxyethylene glycol (PEG) of derivatize, react being enough to form under the condition of hGH-PEG binding substances; With
D) the described hGH-PEG binding substances of purifying.
CNA2005800444637A 2004-12-22 2005-12-21 Methods for expression and purification of recombinant human growth hormone Pending CN101356269A (en)

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