CN1744914A

CN1744914A - Human growth hormone crystals and methods for preparing them

Info

Publication number: CN1744914A
Application number: CN 200380109408
Authority: CN
Inventors: C·戈夫达恩; N·卡拉夫; B·P·西米恩
Original assignee: Altus Biologics Inc
Current assignee: Altus Biologics Inc
Priority date: 2002-12-31
Filing date: 2003-12-31
Publication date: 2006-03-08
Also published as: CN101659701B; CN101659701A; ZA200505305B

Abstract

The present invention relates to stable, extended release crystals of human growth hormone or a human growth hormone derivative and compositions or formulations comprising such crystals. The invention further provides methods for producing those crystals and compositions. The invention further provides methods for treatment of an individual having disorders associated with human growth hormone deficiency or which are ameliorated by treatment with human growth hormone using those crystals and compositions or formulations.

Description

Human growth hormone crystals and their method of preparation

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the crystal and the compositions or the preparation that comprise them of human growth hormone or human growth hormone's derivant.In addition, the invention provides the method for producing human growth hormone or human growth hormone's derivative crystal.Crystal of the present invention has in the mammal of obstacle particularly useful in treatment, described obstacle lacks relevant with the human growth hormone or improves by the personnel selection growth hormone therapy.

The background of invention

Growth hormone or growth hormone (" GH ") are a kind of mammalian proteins, and this albumen comprises a class main body of gland by hormonal system in brain, the synthetic and excretory trophic hormone of adenohypophysis.The excretory GH of adenohypophysis and other trophic hormone are regulated the activity of cell in other endocrine gland of whole body and the tissue.Especially, GH is by the somatotroph secretion of adenohypophysis gland, having stimulates liver and other tissue is synthetic and the function of secretion IGF-1, IGF-1 be a kind of albumen of controlling cell division, adjusting metabolism process and with free state exist or with 6 other albumen that is designated as IGFBP-1 to 6 in a protein binding.Secretion process itself is regulated by somatoliberin (promoting GH to discharge) and growth hormone release inhibiting hormone (suppressing GH discharges) antagonism.

Human growth hormone (" hGH ") is significant especially, because he works as important hormone in the adjusting of cell and organ growth and the physiological function in each old and feeble stage.For example, the excessive child of being created in of hGH causes gigantism and causes acromegaly the adult, otherwise, hGH produces deficiency and causes dwarfism [Mauras etc. the child, J.Clin.Endocrinologyand Metabolism, 85 (10), 3653-3660 (2000); Frindik etc., HormoneResearch, 51 (1), 15-19 (1999); Leger etc., J.Clin.Endocrinologyand Metabolism, 83 (10), 3512-3516 (1998)], Turner syndrome (only limitting the women) [Bramswig, Endocrine, 15 (1), 5-13 (2001); Pasquino etal., Hormone Research, 46 (6), 269-272 (1996)] and chronic renal insufficiency [Carroll etc., Trends in Endocrinology and Metabolism, 11 (6), 231-238 (2000); Ueland etc., J.Clin.Endocrinology and Metabolism, 87 (6), 2760-2763 (2002); Simpson etc., Growth Hormone﹠amp; IGFResearch, 12,1-33 (2002)].The adult, the shortage of hGH can influence the metabolic processes of protein, saccharide, lipid, mineral and connective tissue, and can cause atrophy [Mehls and Haas, the Growth Hormone ﹠amp of muscle, bone or skin; IGF Research, Supplement B, S31-S37 (2000); Fine etc., J.Pediatrics, 136 (3), 376-382 (2000); Motoyama etc., Clin.Exp.Nephrology, 2 (2), 162-165 (1998)].With the deficiency of growing is that other hGH of feature lacks and comprises AIDS become thin syndrome [Hirschfeld, Hormone Research, 46,215-221 (1996); Tritos etc., Am.J.Medicine, 105 (1), 44-57 (1998); Mulligan etc., J.Parenteral and Enteral Nutrition, 23 (6), S202-S209 (1999); Torres and Cadman, BioDrugs, 14 (2), 83-91 (2000)] and Pu-Wei syndrome [Ritzen, Hormone Research, 56 (5-6), 208 (2002); Eiholzer etc., Eur.J.Pediatrics, 157 (5), 368-377 (1998)].

Up to now, the therapeutic scheme of people hGH shortage mainly concentrates on the purification hGH that subcutaneous injection is produced by recombinant DNA technology.This therapeutic agent is packaged into the solution in the tube or needs the freeze-dried powder of reconstruct in use.The frequency of injection changes according to the disease of treatment and the commercially available product of use.For example, dwarfism is treated by subcutaneous injection reorganization every day hGH.

Using subcutaneous administration is that proteic inherent instability is necessary in the solution as the quick route of delivery of hGH.This unstability results from the cracking of crucial intramolecular crosslinking on the inner ad-hoc location of proteinic aminoacid sequence, this and destroy in patient's body by cell surface identification and essential three-Wei structure of being associated with it.The cracking of hGH or mechanism of degradation mainly are to be coordinated by the desamidation of the Oxidation of methionine residue when dissolving or asparagicacid residue, therefore make the albumen inactivation.Because this fragility, in the art to stable and work for a long time, and can not only and can pass through other conventional dosage approach by subcutaneous administration, for example oral area, there is demand in hGH compositions skin and intravenous administration or preparation.

Many commercially available hGH products have been developed to attempt to solve this needs.For example, Nutropin Depot ^It is the injectable recombinant human somatropin's (rhGH) of a kind of implantation polyactide-common Acetic acid, hydroxy-, bimol. cyclic ester (PLG) microsphere suspension (referring to http://www.gene.com).Except that rhGH and PLG, microsphere also comprises the composition of zinc acetate and zinc carbonate.Before administration, solid matter must be with the aqueous solution reconstruct that comprises sanlose, Polysorbate, sodium chloride and water.This suspension of mainly being made up of polymer was administered once or also required for twice and injects with the injection needle of 21 gauges in every month.Because disadvantageous injection site reaction can take place in the size of microsphere and the viscosity of product, cause brief summary, erythema, pain, blue or green midship, scratch where it itches, lipoatrophy and swelling (referring to Http:// www.genentech.com/Gene/products/information/opportunistic/nutropin-depot/i ndex.jsp).

Another under development but suspended again subsequently hGH product is Albutropin ^TM, a kind of by the human albumin that works for a long time of genetic method production and human growth hormone's fusion rotein (referring to http://www.hgsi.com/products/albutropin.html).It is said that this product shows the half-life of prolongation in circulation, increase about 50% than half-life of soluble natural hGH.Albutropin ^TMDrug administration by injection on basis weekly, and the level that after by removing for a long time in health, stimulates IGF-1 typically.The biological effect of this product and current available growth hormone therapy are similar.

Another product that has been developed is Infitropin CR ^TM, a kind of hGH preparation that constitutes by the hGH molecule of Polyethylene Glycol-put together.This hGH that puts together requires jede Woche to inject once and it is said with successive speed release not tangible burst effect [Ross etc., J.Biol.Chem., 271 (36), 21696-21977 (1996)].Yet this product is stopped.

United States Patent (USP) 5,981,485 and 6,448,225 mention the water formulation of hGH, said preparation allegedly do not need reconstruction step and every day drug administration by injection.This preparation comprises hGH typically, buffer, nonionic surfactant and optionally, neutral salt, mannitol, perhaps antiseptic.

Other medicines medicine-feeding technology miscellaneous, hydrogel [Katakam etc., J.Controlled Release, 49 (1) for example, 21-26 (1997)], liposome, oil emulsion and biodegradable polymer microsphere, the hGH that has been used to attempt to provide lasting discharges.Yet the preparation that obtains shows the drug release of burst, and using exacting terms and wherein some is complicated to making.These are especially real for the hGH preparation that is total to hydroxyacetic acid (PLGA) microsphere technology based on DL-lactic acid, because be used to produce temperature, surfactant, organic solvent, He Shui/organic solvent interface that the method trend of microsphere uses some conditions for example to improve, all these can cause albuminous degeneration [Herberger etc., Proc.Intl.Symp.Controlled Release of Bioactive Materials, 23,835-836 (1996); Kim etc., Intl.J.Pharmaceutics, 229 (1-2), 107-116 (2001)].

More above-described preparations require hGH to be stored in lyophilised state, and this may be time-consuming and an expensive process.United States Patent (USP) 5,780,599 and 6,117,984 relate to the bivalent cation crystallization of hGH and produce the crystalline method of hGH bivalent cation, do not need the lyophilization step.

Although effort solves the shortcoming of the hGH product of routine, comprise interior half-life of unstability, body short, happen suddenly effect, shortage oral administration biaavailability and administration difficulty and administration frequency when preserving and injecting, but still the needs of existence improvement hGH preparation.In order to address that need, the present invention advantageously provides the human growth hormone's who produces hGH stable, that work for a long time crystallization.

Summary of the invention

The present invention relates to stable, that work for a long time, easily with patient close friend's the human growth hormone or the crystal of human growth hormone's derivant.The present invention provides the crystalline compositions of human growth hormone or human growth hormone's derivant further, comprises the acceptable composition of its medicine.The present invention provides preparation this crystalloid further, and the method for compositions that comprises them.Crystal of the present invention and compositions are advantageously used in the method that treatment has the individuality of obstacle, and described obstacle lacks relevant with the human growth hormone or improves by the personnel selection growth hormone therapy.

The crystal of human growth hormone or human growth hormone's derivant, the compositions or the preparation that perhaps comprise them, several advantages are arranged, comprise: can be administered once available immediately crystalline form of suspension, safety weekly, effectiveness, purity, stability, resuspending and syringeability in short time period.In view of the application's disclosure, it should be appreciated by those skilled in the art that other purpose of the present invention, comprise with the hGH preparation of routine and comparing, hGH crystal and comprise their compositions or the improvement of preparation.

The simple declaration of accompanying drawing

Fig. 1 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 860mM ammonium phosphate (pH8.9).See embodiment 1.

Fig. 2 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of the 390mM sodium citrate.See embodiment 2.

Fig. 3 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 600mM disodium hydrogen phosphate and 100mM Tris-HCl (pH8.6).See embodiment 3.

Fig. 4 illustrated as by the optical microscopy imaging, in the presence of 85mM calcium acetate and 100mMTris-HCl (pH8.6) the hGH crystal growth and with protamine sulfate (1mg/ml) cocrystallization.See embodiment 4.

Fig. 5 shown as the function of time and in 280nm monitoring, the crystalline dissolubility of hGH that generates from ammonium phosphate, sodium citrate, disodium hydrogen phosphate and calcium acetate/protamine precipitant.See embodiment 5.

Fig. 6 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 10% (v/v) isopropyl alcohol, 85mM calcium acetate and 100mM Tris-HCl (pH8.6).See embodiment 6.

Fig. 7 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 5% (v/v) isopropyl alcohol, 85mM calcium chloride and 100mM Tris-HCl (pH8.6).See embodiment 7.

Fig. 8 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 10% (v/v) ethanol, 10% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6).See embodiment 8.

Fig. 9 shown as the function with time of being divided into unit, in the 280nm monitoring, and the hGH crystalline dissolubility that generates according to embodiment 6-8.See embodiment 9.

Figure 10 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 85mM calcium acetate, 2% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6).See embodiment 10.

Figure 11 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 500mM sodium acetate, 6% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6).See embodiment 11.

Figure 12 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 85mM calcium chloride, 6% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6).See embodiment 12.

Figure 13 illustrated as by the optical microscopy imaging, in the presence of 85mM calcium acetate, 6% (v/v) PEG-6000,100mM Tris-HCl (pH8.6) the hGH crystal growth and with protamine sulfate (1mg/ml) cocrystallization.See embodiment 13.

Figure 14 has illustrated as passing through optical microscopy imaging, hGH crystal growth in the presence of 125mM calcium acetate, 6% (v/v) PEG-MME-6000 and 100mM Tris-HCl (pH8.6).See embodiment 14.

Figure 15 shown as the function with time of being divided into unit, in the 280nm monitoring, and the hGH crystalline dissolubility that generates according to embodiment 10-14.See embodiment 15.

Figure 16 has shown coml hGH (soluble hGH) and the serum levels (ng/ml) of hGH (hGH crystal) in female Sprague-Dawley rat for preparing according to embodiment 10, every rat single is subcutaneous give the soluble of 2.5mg/kg dosage or crystallization hGH after, sampling in 24 hours.At t=0,0.5,1,2,4,6,8,12 and 24 hours mensuration serum levels.See embodiment 16.

Figure 17 has illustrated the dissolving characteristic of the hGH crystal (forming) after adding not commensurability protamine sulfate in the presence of 85mM calcium acetate, 2% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6).The crystalline preparation of then that these are different hGH adds in the dissolving buffer and is preceding in the concentration (area) of measuring solvable hGH in the supernatant by RP-HPLC, allows to place 1 hour.See embodiment 17.

Figure 18 A has shown as passing through the imaging of TEM lengthwise, hGH crystal growth in the presence of 500mM sodium acetate, 6%v/vPEG-6000 and 100mM Tris-HCl (pH8.6).See embodiment 18.

Figure 18 B has illustrated as passing through the imaging of TEM cross section, hGH crystal growth in the presence of 500mM sodium acetate, 6%v/vPEG-6000 and 100mM Tris-HCl (pH8.6).See embodiment 18.

Figure 19 A has shown the serum levels (ng/ml) of hGH in the female Sprague-Dawley rat of group 3-5 and 9, in every rat seven days every day subcutaneous giving or sampling (0-72 of demonstration hour) in 168 hours behind the hGH of the subcutaneous 6.7mg/kg of the giving dosage of single in seven days.See embodiment 22 and table 7-12.

Figure 19 B has shown subcutaneous giving every day (group 2) in every rat seven days or in 8 days, selected the weight increase of the female Sprague-Dawley rat of preparation (group 2,4 and 9) behind the hGH of first day subcutaneous giving of single (group 4 and 9) 6.7mg/kg dosage of seven days.See embodiment 22 and table 13.

Figure 20 A has shown the concentration of female young macaque as hGH in the serum of the function of time, according to table 16, described monkey every day is subcutaneous give soluble hGH (group 1), with the hGH sodium crystal (group 2) of poly arginine complexation and with the hGH sodium crystal (group 3) of protamine complexation.See embodiment 23.

Figure 20 B has shown the concentration of female young macaque as IGF-1 in the serum of the function of time, according to table 18, described monkey every day is subcutaneous give soluble hGH (group 1), with the hGH sodium crystal (group 2) of poly arginine complexation and with the hGH sodium crystal (group 3) of protamine complexation.See embodiment 23.

Figure 21 A has shown the concentration of female young macaque as hGH in the serum of the function of time, according to table 20, described monkey by every day subcutaneous give soluble hGH (group 1), with the hGH sodium crystal of protamine complexation (hGH of 3: 1 ratios: protamine) (group 2) and with the hGH sodium crystal (hGH of 2: 1 ratios: protamine) (group 3) of protamine complexation.See embodiment 24.

Figure 21 B has shown the concentration of female young macaque as IGF-1 in the serum of the function of time, according to table 22, described monkey by every day subcutaneous give soluble hGH (group 1), with the hGH sodium crystal of protamine complexation (hGH of 3: 1 ratios: protamine) (group 2) and with the hGH sodium crystal (hGH of 2: 1 ratios: protamine) (group 3) of protamine complexation.See embodiment 24.

Figure 22 has illustrated growths in seven days of male Wistar rat, according to table 25, described rat is by subcutaneous contrast the (in organizing 1, seven day once a day), soluble hGH (group 4 and 5, in seven days once a day) and crystal hGH (in organizing 6,7,9 and 10, seven days once).See embodiment 25.

Figure 23 has illustrated the weight increase (gram) of inductive male Wistar rat every day in seven days, according to table 26, described rat is by subcutaneous contrast the (group 1, in seven days once a day), soluble hGH (group 4 and 5, in seven days once a day) and crystal hGH (in organizing 6,7,9 and 10, seven days once).See embodiment 25.

Detailed description of the present invention

Definition

Unless other definition is arranged herein, relevant with the present invention use the Science and Technology term should have the general implication of understanding of those of ordinary skills. In addition, unless contextual other requirement, single term should comprise the term of plural number and the term of plural number should comprise single term. In general, be well-known and normally used in the art with column chromatography described herein, optical microscopy, UV-VIS spectroscopy, pharmacokinetic analysis, recombinant DNA method, peptides and proteins buzz word and technology chemical, that nucleic acid chemistry is relevant with molecular biology.

Following term except as otherwise noted, should be understood that to have following meanings:

Term " growth hormone (GH) " is often referred to the excretory growth hormone of mammal pituitary gland.Although the tabulation of neither one limit, mammiferous example comprises people, ape, monkey, rat, pig, Canis familiaris L., rabbit, cat, cow, horse, mice, rat and goat.According to an embodiment preferred of the present invention, mammal is the people.

" human growth hormone (hGH) " represents a kind of have natural human growth hormone's aminoacid sequence, the protein of 26S Proteasome Structure and Function feature.As used herein, human growth hormone (hGH) also comprises any isotype of natural human growth hormone, includes but not limited to, molecular mass is 5,17,20,22,24,36 and isotype [the Haro etc. of 45kDa, J.Chromatography B, 720,39-47 (1998)].Like this, term hGH comprises 191 aminoacid sequences of natural hGH (growth hormone) and comprises 192 aminoacid sequences (Met-hGH) and the somatrem [United States Patent (USP) 4,342,832 and 5,633,352] of N-terminal methionine.HGH also can be by obtaining from the biogenetic derivation separation and purification or by recombinant DNA method.If make by recombinant DNA method, hGH is named as recombinant human somatropin (rhGH).Met-hGH prepares by recombinant DNA method typically.

Term " human growth hormone's derivant " refers to have and the protein of the comparable aminoacid sequence of human growth hormone of existence naturally.Term " comparable " refer to and 192 aminoacid sequences of 191 aminoacid sequences of hGH or Met-hGH have from 2% to 100% homologous aminoacid sequence.In different embodiments of the present invention; human growth hormone's derivant comprises the organic cation of hGH or Met-hGH; the proteinic displacement of biosynthetic hGH or Met-hGH; disappearance and the variant that inserts; the hGH of post translational modification or Met-hGH protein; comprise desamidation; phosphorylation; glycosylation (glycoslylation); acetylation; polymerization and enzymatic lysis reaction [Haro etc.; J.Chromatography B; 720; 39-47 (1998)], the hGH that comes from biogenetic derivation of chemical modification or Met-hGH protein; the polypeptide analog and the chemical synthesising peptide that comprise the aminoacid sequence that is similar to hGH or Met-hGH.

The method that is used for preparing hGH or Met-hGH comprises from biogenetic derivation separation, recombinant DNA method, synthetic chemistry approach or their combination.Up to now, the gene of the different DNA sequence of coding hGH comprises hGH-N and hGH-V[Haro etc., J.Chromatography B, 720,39-47 (1998); Bennani-Baiti etc., Genomics, 29,647-652 (1995)].

Term " valency " is defined as a kind of element and the bonded ability of other element, it is by in the decision of the number of electrons of atom outermost shell and be expressed as can be in conjunction with the atomic number [Webster ' s New WorldDictionary of Science of the hydrogen (perhaps any other standard monogen) of (or substituting) its atom, Lindley, D.and Moore T.H., Eds., Macmillan, New York, New York, 1998].Term " monovalent cation " and " bivalent cation " refer to have respectively the ion that has positive charge of monovalence state or bivalence state.Cation with different price state can be organic or inorganic at nature.The example of monovalence inorganic cation comprises ammonium (NH ⁴⁺) and periodic chart group I element (H ⁺, Li ⁺, Na ⁺, K ⁺, Rb ⁺, Cs ⁺And Fr ⁺), and the bivalence inorganic cation comprises group II element (Be ⁺, Mg ²⁺, Ca ²⁺, Sr ²⁺, Ba ²⁺, Mn ²⁺, Co ²⁺, Ni ²⁺, Cu ²⁺, Zn ²⁺, Cd ²⁺, MO ²⁺And Ra ²⁺).

" the calcium crystal of human growth hormone or human growth hormone's derivant " refers to when the divalent calcium ion exists by crystalline human growth hormone or its derivant.The divalent calcium ion is imported into crystallization solution with the form of calcium salt.In preferred embodiments, the calcium crystal of human growth hormone or human growth hormone's derivant comprises the monomer of everyone growth hormone or human growth hormone's derivant or monomer chain about 1 to about 500 calcium molecules.In a preferred embodiment, the calcium crystal of human growth hormone or human growth hormone's derivant comprises the monomer of everyone growth hormone or human growth hormone's derivant or monomer chain about 1 to about 140 calcium molecular compositions.

Term " calcium salt " comprises with calcium ion forms the inorganic of ionic bond and organically gegenion or molecule.The example of different calcium salts comprises the calcium acetate hydrate, the calcium acetate monohydrate, the acetylacetonate hydrate of calcium, L-ascorbic acid calcium dihydrate, two (6,6,7,7,8,8,8-fluoro-in heptan 2,2-dimethyl-3, the 5-acetyl caproylization) calcium, two (2,2,6,6-tetramethyl-3, the 5-heptadioneization) calcium, calcium bromide, calcium carbonate, calcium chloride, calcium chloride dihydrate, calcium chloride hexahydrate, calcium chloride hydrate, the calcium citrate tetrahydrate, dalcium biphosphate, 2 ethyl hexanoic acid calcium, calcium fluoride, calcium gluconate, calcium hydroxide, calcium hypochlorite, calcium iodate, calcium iodide, the calcium iodide hydrate, ionophore I calcium, calcium molybdate, lime nitrate, calcium oxalate, the calcium oxalate hydrate, calcium oxide, calcium pantothenate, calcium propionate, calcium pyrophosphate and calcium sulfate.In an embodiment preferred of the present invention, calcium salt is selected from the group of being made up of calcium acetate, calcium chloride, calcium sulfate and calcium gluconate.In a preferred embodiment, calcium salt is a calcium acetate.

" the organic cation crystal of human growth hormone or human growth hormone's derivant " refers to when organic cation exists by crystalline human growth hormone.Term " organic cation " refers to positively charged atom or wraps the carbonaceous former group of giving.The organic cations example comprises quaternary ammonium cation, etamon (TEA), tributyl-methyl phosphonium ammonium (TBuMA), procainamide ethobromide (PAEB), azido procainamide N-methiodide (APM), d tubocurarine, diformazan curare vecuronium bromide (metocurine vecuronium), Rocuronium Bromide, 1-methyl-4-phenylpyridinium, choline and N-, and (4, the 4-axo-n-pentyl)-21-deoxidation ajmaline (ajmalinium) (APDA).

HGH is commercial can lyophilized form to be obtained and produces by recombinant DNA method typically.According to the present invention, the crystallization of hGH is buffer solution, purification and/or desalination, dialysis and the concentrated solution by preparation hGH and add monovalence or bivalent cation or salt finish in solution usually.The step of back causes the cationic formation of bonded hGH organic or inorganic.

An embodiment preferred of the present invention relates to the monovalent cation crystal of hGH or hGH derivant.In a preferred embodiment, monovalent cation is selected from the group of being made up of lithium, sodium, potassium and ammonium.In a most preferred embodiment, monovalent cation is a sodium.In a most preferred embodiment, human growth hormone or human growth hormone's derivant comprise the monomer of everyone growth hormone or human growth hormone's derivant or monomer chain approximately from 1 to about 500 monovalent cation molecules.

Term " monovalent cation salt " comprises with monovalent ion forms the inorganic of ionic bond and organically gegenion or molecule.In a preferred embodiment, monovalent cation salt is sodium salt.In a preferred embodiment, sodium salt is selected from the group of being made up of sodium citrate, sodium phosphate and sodium acetate.In a most preferred embodiment, sodium salt is a sodium acetate.

Another embodiment preferred of the present invention relates to the protamine crystal of hGH or hGH derivant.Similarly, also in another preferred embodiment, the present invention relates to poly arginine (polyarginine) crystal of hGH or hGH derivant.

Another one embodiment preferred of the present invention comprises with protamine or poly arginine and combining or the hGH of cocrystallization or the monovalence or the bivalence crystal of hGH derivant.More preferably, crystal is the sodium crystal with protamine or poly arginine complexation or cocrystallization.

The soluble form of hGH can characterize with several different methods, comprise reversed phase high-performance liquid chromatography (RP-HPLC), size exclusion chromatography high performance liquid chromatography (SEC-HPLC) and hydrophobic interaction chromatography (HIC) [Wu etc., J.Chromatography, 500,595-606 (1990); " Hormone Drugs ", FDA publication, (1982)].On the other hand, the crystalline form of hGH can characterize with optical microscopy and X-ray diffraction.Generally speaking, crystallization condition will determine the shape of albumin crystal, just, be selected from the shape by spherical, needle-like, bar-shaped, plate-like (hexagonal and foursquare), rhombohedral, cube, bicone and the prismatic group of forming.

According to the present invention, when with the optics microscopy imaging, the crystal formation bar-shaped or acicular form of hGH or hGH derivant.In one embodiment, the bar-shaped or needle-like of the crystal formation length of hGH or hGH derivant between about 0.1 to about 200 μ m.In a preferred embodiment, the bar-shaped or needle-like of the crystal formation length of hGH or hGH derivant between about 3 to about 100 μ m.In a preferred embodiment, the bar-shaped or needle-like of the crystal formation length of hGH or hGH derivant between about 10 to about 25 μ m.

Another embodiment of the invention relates to the compositions of the calcium, monovalent cation, protamine or poly arginine crystal and the pharmaceutically acceptable excipient that comprise hGH or hGH derivant.Also in another preferred embodiment, about 1: 250 hGH to about 1: 20 molar ratio of the crystal of hGH or hGH derivant and excipient: excipient is present in such compositions.In another selectable embodiment preferred, the crystal of hGH or hGH derivant and excipient are to arrive the hGH of about 1: 10 molar ratio in about 3: 1: excipient exists.In going back another embodiment preferred, the crystal of hGH or hGH derivant and excipient are to arrive the hGH of about 1: 0.125 molar ratio in about 1: 10: excipient exists.In a preferred embodiment, the crystal of hGH or hGH derivant is grown with sodium acetate, and it may or wrap quilt with poly arginine or protamine crystallization.

The crystal of human growth hormone or human growth hormone's derivant can combine with any pharmaceutically acceptable excipient.According to the present invention, " pharmaceutically acceptable excipient " is the excipient that plays filler or fill composition effect that uses in pharmaceutical composition.The preferred excipient that is included in this classification is: 1) amino acids, for example glycine, arginine, aspartic acid, glutamic acid, lysine, agedoite, glutamine, proline; 2) saccharide, for example, monosaccharide is glucose, fructose, galactose, mannose, arabinose, xylose, ribose for example; 3) disaccharides, for example lactose, trehalose, maltose, sucrose; 4) polysaccharide, for example maltodextrin, glucosan, starch, glycogen; 5) acyclic polyhydric alcohol class, for example mannitol, xylitol, lactose, Sorbitol; 6) glucuronic acid, galacturonic acid; 7) cyclodextrin, for example Methyl flamprop, HP-and analog; 8) inorganic molecule class, for example phosphate of sodium chloride, potassium chloride, magnesium chloride, sodium and potassium, boric acid, ammonium carbonate and ammonium phosphate; 9) organic molecule class, for example acetate, citrate, Ascorbate, lactate; 10) for example arabic gum, diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol, poloxamer, Polysorbate, sodium lauryl sulphate, stearic acid, sorbitan monolaurate, anhydrosorbitol monostearate and other anhydrosorbitol derivant, poly-oxyl derivant, wax, polyethylene oxide derivatives, anhydrosorbitol derivant of emulsifying or solubilising/stabilizing agent; With 11) for example increase viscous agent, agar, alginic acid and its salt, guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivant, propylene carbonate, Polyethylene Glycol, hexanediol, tyloxapol.The salt of these chemical compounds also can be used.Preferred one group of excipient comprises salt, for example acetate, phosphate, citrate and borate, glycine, arginine, polyethylene oxide, polyvinyl alcohol, Polyethylene Glycol, hexanediol, methoxy poly (ethylene glycol), gelatin, HP-, polylysine and the poly arginine of sucrose, trehalose, lactose, Sorbitol, lactose, mannitol, inositol, sodium and potassium.

In one embodiment of the invention, excipient is selected from the group of being made up of aminoacid, salt, alcohol, saccharide, protein, lipid, surfactant, polymer, polyamino acid and their mixture.In a preferred embodiment, excipient is selected from the group of being made up of protamine, polyvinyl alcohol, cyclodextrin, glucosan, calcium gluconate, polyamino acid, for example poly arginine, polylysine and polyglutamic acid, Polyethylene Glycol, dendrimers, poly ornithine (polyorthinine), polymine, chitosan and their mixture.In a preferred embodiment, excipient is selected from the group of being made up of protamine, poly arginine, Polyethylene Glycol and their mixture.

According to the present invention, the crystal of human growth hormone or human growth hormone's derivant also can combine with carrier or excipient, and this carrier or excipient are a kind of when it is added a kind of therapeutic agent, can quicken or improve the material of the effect of therapeutic agent.[The On-Line MedicalDictionary，http：//cancerweb.ncl.ac.uk/omd/index.html]。The example of carrier or excipient comprises, for example, the material and the Polyethylene Glycol of partial glycerol ester admixture, water, salt or the electrolyte of buffer material, for example phosphate, glycine, sorbic acid, potassium sorbate, saturated vegetable fatty acid, for example protamine sulfate, sodium hydrogen phosphate, sodium chloride, zinc salt, colloid silicon, magnesium, trisilicate, cellulose base.The carrier of gel-type vehicle form or excipient can comprise, for example, and sodium carboxymethyl cellulose, polyacrylate, polyethylene glycol oxide-polyoxypropylene block copolymers, Polyethylene Glycol and lignoceryl alcohol (wood wax alcohols).

In going back a preferred embodiment, excipient is a protamine.And the crystal of hGH or hGH derivant and protamine are with the hGH of about 5: 1 to about 1: 10 (w/w) ratios: protamine exists.This ratio also can be between about 10: 1 to about 20: 1 (w/w).More preferably, this ratio is between about 12: 1 to about 15: 1 (w/w).According to a selectable embodiment, this ratio is between about 3: 1 to about 1: 10 (w/w).In another embodiment, this ratio is between about 5: 1 to about 40: 1 (w/w).And in further embodiment, this ratio is about 5: 1 (w/w).

In another aspect of this invention, pharmaceutically acceptable excipient is selected from by polyamino acid, comprises the group that polylysine, poly arginine and polyglutamic acid are formed.In an embodiment preferred of the present invention, excipient is a polylysine.In a preferred embodiment, the molecular weight of polylysine is about 1,500 and about 8, between the 000kD.In another embodiment, the crystal of hGH or hGH derivant and polylysine are with (w/w) hGH of about 5: 1 to about 40: 1 ratios: polylysine exists.This ratio also can be between about 10: 1 to about 20: 1 (w/w).More preferably, this ratio is between about 12: 1 to about 15: 1 (w/w).According to a selectable embodiment, this ratio is between about 5: 1 to about 1: 50 (w/w).And in further embodiment, this ratio is about 5: 1 (w/w).

In also another embodiment preferred of the present invention, excipient is a poly arginine.In a preferred embodiment, the molecular weight of poly arginine is about 15,000 and about 60, between the 000kD.In another embodiment, the crystal of hGH or hGH derivant and poly arginine are with the hGH of about 5: 1 to about 40: 1 (w/w) ratios: poly arginine exists.This ratio also can be between about 10: 1 to about 20: 1 (w/w).More preferably, this ratio is between about 12: 1 to about 3: 1 (w/w).According to a selectable embodiment, this ratio is between about 5: 1 to about 1: 50 (w/w).In another embodiment, this ratio was about 12: 1 scopes to about 15: 1 (w/w).And in further embodiment, this ratio is about 5: 1 (w/w).

Comprise the crystalline a kind of injectable crystalline suspension that contains about 20mg/ml hGH or hGH derivant according to one embodiment of the invention.This suspension is characterized as easy resuspending, slow falling shallow lake and is approximately 7 days time effect feature.It can be injected weekly once, with the syringe of 30 gauges and the payload of 80% level is provided.As gather the reflection of (SE-HPLC) and 2.3% related protein (RP-HPLC) by parameter 0.02%, this suspension is pure.This purity was kept 4 months under the condition of cold preservation at least.

One embodiment of the invention relate to the crystal of hGH or hGH derivant, compare with the solvable hGH or the hGH preparation of routine, and being characterized as of they has dissolving delay behavior when inserting individuality.According to the present invention, the crystalline dissolving characteristic of hGH or hGH derivant is in the body or external solubility parameter.For example, dissolution in vitro is described as in a successive course of dissolution concentration of per 15 minutes or the resulting solvable hGH of each washing step (being expressed as the crystalline percentage ratio of total hGH of initial existence or hGH derivant or total hGH or the crystalline mg of hGH derivant) (referring to embodiment 5).In one embodiment of the invention, the crystalline feature of hGH or hGH derivant shows as and be exposed to dissolving buffer (50mM HEPES (pH7.2), 140mM NaCl, 10mM KCl and 0.02% (v/v) NaN when 37 ℃ of temperature ₃) time, the said crystalline dissolution in vitro rate of each washing step about 2 to about 16%, wherein the concentration of hGH or hGH derivant exists with about 2mg/ml in the solution.In another embodiment, the crystalline feature of hGH or hGH derivant shows as in a successive course of dissolution, the said crystalline dissolution in vitro rate (referring to embodiment 5) of each washing step about 0.04 to about 0.32mg.On the other hand, after going into mammal at single injection hGH, in time hGH serum levels is described dissolving in the body in the mammalian body.

In mammalian body, GH stimulates the synthetic and secretion IGF-1 of tissue, a kind of protein that works successively in cell division and metabolic processes.Just as the skilled person will appreciate, serum hGH and IGF-1 level depend on many factors, comprise the physiological factor relevant with treatment.Such factor comprises, but be not limited to: physiologic factor, for example: birth age and bone age, sex, body weight, stage of development (for example, the level that increases in adolescence) factor relevant, for example speed of dosage, administration (kinetics) and route of administration with treatment.Similarly, it will be understood by those skilled in the art that hGH and the IGF-1 levels different for different patient crowds also may be useful from the position of safety and effect.

Suffering from multiple hGH shortage, morbid state or syndromic adult or child can treat by the scheme of the multiple exogenous hGH that sends by using the crystal according to hGH crystal of the present invention or hGH derivant.For example, the endocrinologist can be with the dosage in about 0.2mg/kg/ week to child's begin treatment, increases dosage later on to 0.3mg/kg/ week through the treatment of several weeks or some months, and about adolescence, dosage can further be increased to about 0.7mg/kg/ week.Just as the skilled person will appreciate, this exogenous level to needing adult that hGH sends or child to send hGH of sending also depends on physiological level or the concentration that hGH exists.

Adult and child's hGH dosage is represented with mg/kg or iu (IU/kg).Such scheme is usually by being ranked in sky or week, just, and mg/kg/ days or mg/kg/ week.This consideration is arranged, according to one embodiment of the invention, the crystal of hGH or hGH derivant, or comprise the single-dose of these crystalline compositionss, for example, every 30kg child is the about 9mg of single-dose weekly, provides in the body hGH serum-concentration greater than about 10ng/ml after administration on the the 1st and the 2nd day, after administration in the 3rd and the 4th day body the hGH serum-concentration greater than about 5ng/ml and after administration the about 0.3ng/ml of hGH serum-concentration in the 5th and the 7th day body.Selectively, the crystal of hGH or hGH derivant, or comprise the single-dose of these crystalline compositionss, in said mammal, provide about 0.3ng/ml to about 2,500ng/ml hGH, preferably approximately 0.5ng/ml is to about 1,000ng/ml hGH, more preferably about hGH serum-concentration in the body from 1ng/ml to about 100ng/ml hGH reaches after the administration about 0.5 hour to about 40 days, preferably each in about 0.5 hour to about 10 days, 7 days or 1 day after the administration.Similarly, the crystal of hGH or hGH derivant, or comprise the single-dose of these crystalline compositionss, in said mammal, provide greater than about 2ng/ml hGH, be preferably greater than about 5ng/ml hGH, more preferably greater than serum-concentration in the body of about 10ng/ml hGH, reach after the administration about 0.5 hour to about 40 days, each after the preferred administration in about 10 days, 7 days or 1 day.In a preferred embodiment of the present invention, the crystal of hGH or hGH derivant, or comprise the single-dose of these crystalline compositionss, in said mammal, provide greater than serum-concentration in the body of about 0.3ng/ml hGH, reach after the administration about 0.5 hour to about 40 days, arbitrary stage of each after the preferred administration in 10 days, 7 days or 1 day.According to one embodiment of the invention, the crystal of hGH or hGH derivant, or comprise the single-dose of these crystalline compositionss, after administration, provided in the body hGH serum-concentration greater than about 10ng/ml on the the 1st and the 2nd day, after administration in the 3rd and the 4th day body the hGH serum-concentration greater than about 5ng/ml and after administration in the 5th and the 7th day body the hGH serum-concentration greater than about 0.3ng/ml.And in further embodiment, the crystal of single-dose hGH or hGH derivant, or comprise this crystal-like compositions provides greater than serum-concentration in the body of about 0.3ng/ml hGH, reaches after the administration about 0.5 hour to about 10 days.

According to one embodiment of the invention, the crystal of single-dose hGH or hGH derivant, or comprise this crystal-like compositions, IGF-1 serum rising in the body is provided, this serum raises more than the baseline IGF-1 level before described using, from using back about 10 hours to about 72 hours greater than about 50ng/ml, and from using back about 72 hours to about 15 days, preferably used the back about 10 days, at about 0.5ng/ml extremely between about 50ng/ml.Alternately, the crystal of single-dose hGH or hGH derivant, or comprise this crystal-like compositions, provide about 5ng/ml to about 2,500ng/ml, preferably about 100ng/ml extremely about 1, IGF-1 serum raises in the body of 000ng/ml, reaches to use the back about 0.5 hour to about 40 days, preferably uses the back about 7 days.Alternately, the crystal of single-dose hGH or hGH derivant, or comprise this crystal-like compositions, can provide more than about 50ng/ml according to the present invention, the above interior IGF-1 serum of body of preferred about 100ng/ml raises, and reaches and uses the back about 0.5 hour to about 40 days, preferably uses the back about 7 days.According to one embodiment of the invention, the crystal of single-dose hGH or hGH derivant, or comprise this crystal-like compositions, IGF-1 serum rising in the body is provided, more than the baseline IGF-1 level of this serum rising before described using, from using back about 10 hours to about 72 hours, and from using the back about 72 hours to about 15 days or using afterwards about 72 hours to about 10 days, at about 0.5ng/ml extremely between about 50ng/ml greater than about 50ng/ml.

According to the present invention, single-dose is defined as thoughtful about 100mg/kg/ week hGH crystal of about 0.01mg/kg/ or hGH derivative crystal, or comprises this crystal-like compositions, wherein the volume of administration is between 0.1ml and about 1.5ml.For example, can be with about 0.3mg/kg/ week, for example about 9mg for the 30kg child with hGH crystal or hGH derivative crystal, or comprises this crystal-like compositions administration children growth hormonoprivia.Can be with about 0.375mg/kg/ week, for example about 11.25mg for the 30kg child with hGH crystal or hGH derivative crystal, or comprises this crystal-like compositions administration Turner syndrome.Additionally, can be with about 0.2mg/kg/ week, for example about 16mg for the 80kg adult uses hGH administration adult growth hormone deficiency.Can be with 6mg/ days, 42mg/ week for example is with the hGH administration AIDS disease of becoming thin.

Go back in the embodiment of the present invention, hGH crystal or hGH derivative crystal, or comprise this crystal-like compositions, in mammal, show and the similar relative bioavailability of soluble hGH.Compare with the relative bioavailability of the solvable hGH that sends by identical approach, have at least 50% or bigger relative bioavailability according to crystal of the present invention, wherein, measure described bioavailability by the area under curve (AUC) of hGH serum-concentration in total body for described soluble hGH and described crystal.Therefore the crystal of hGH or hGH derivant is a feature with rate of dissolution in the favourable body.

The present invention further provides the crystalline method that the mammal with obstacle is given hGH or hGH derivant, described obstacle lacks relevant with the human growth hormone or or by improving with hGH treatment.Described method comprises the step of mammal being treated the hGH or the hGH derivative crystal of effective dose.Alternately, described method comprises and comprises hGH or hGH derivative crystal separately or have a step of the compositions of excipient to what mammal was treated effective dose.Different embodiments according to hGH of the present invention or hGH derivative crystal is: the calcium crystal of hGH or hGH derivant, monovalence crystal, protamine crystal or poly arginine crystal.Can by per three days approximately once, a week approximately once, per two weeks approximately once or every month time scheme approximately once give such crystal, or comprise their compositions.

Can lacking relevant obstacle with hGH and include, but are not limited to: adult's growth hormone deficiency, children growth hormonoprivia, Pu-Wei syndrome, Turner syndrome, short bowel syndrome, chronic renal insufficiency, spontaneous short stature, dwarfism, hypopituitarism dwarfism, osteanagenesis, female sterile disease, intrauterine growth retardation, AIDS related cachexia, segmental enteritis, burn and other heredity and dysbolismus according to the present invention treatment.In one embodiment of the invention, described obstacle is a children growth hormonoprivia and in the child of experience treatment, treatment causes the annual speed of growth between about 7cm and the about 11cm.

In another embodiment of the invention, the calcium crystal of hGH or hGH derivant can be used as bone-specific drug in mammal, and the useful adjuvant of the treatment that lacks of human growth hormone and working.

The present invention also is provided at the method for inducing weight increase in the mammal, and this method comprises treats the hGH of effective dose or the crystalline step of hGH derivant to described mammal.Alternately, these class methods comprise the step of described mammal being treated the compositions of the crystal that comprises hGH or hGH derivant of effective dose and excipient.In an embodiment of these class methods, after giving described crystal by injection weekly, inductive weight increase is between about 5% and about 40% in the rat that hypophysectomizes.

The crystal of the crystal of hGH, hGH derivant or comprise that they are independent, or have the compositions of excipient can be independent, or be given as the part of medicine, treatment or prevention preparation.Can comprise that for example, parenteral, mouth, lung, nose, ear, anus, skin, eye, intravenous, intramuscular, intra-arterial, intraperitoneal, mucosa, Sublingual, subcutaneous, transdermal, part, cheek or intracranial approach give them by any conventional route of administration.

In one embodiment of the invention, through port approach or parenteral approach give the crystal of hGH or hGH derivant, or comprise their compositions, are with or without excipient.In preferred embodiments, give the crystal of hGH or hGH derivant by subcutaneous or intramuscular approach, or comprise their compositions, be with or without excipient.

In preferred embodiments, use the pin have more than or equal to 27 gauge, give crystal of the present invention or compositions by subcutaneous route.In one embodiment of the invention, described pin gauge can equal 30.Can give described crystal or compositions from the syringe or the change dosage infusion pump of pre-filling.Perhaps, give them by Needleless injection.

The present invention advantageously allows hGH to continue to be discharged in the mammal.In one embodiment, a week once gives approximately according to crystal of the present invention or compositions.In another embodiment, per two weeks once give approximately according to crystal of the present invention or compositions.In going back another embodiment, once gave according to crystal of the present invention or compositions approximately in every month.It will be apparent to one skilled in the art that the concrete therapeutic scheme disease that for example will treat that will depend on factor, the patient's age that will treat and body weight, patient's general physical condition and treatment doctor's judgement.

According to an embodiment, the compositions that comprises hGH of the present invention or hGH derivative crystal is a feature with the hGH concentration greater than about 0.1mg/ml.For example, described concentration can be between about 0.1mg/ml and about 100mg/ml.Perhaps, those compositionss may with between about 1mg/ml and the about 100mg/ml or the hGH concentration between about 10mg/ml and the about 100mg/ml be feature.Such compositions also comprises following component: mannitol-about 0.5mg/ml is to about 100mg/ml; Sodium acetate-about 5mM is to about 250mM (preferably about 25mM is to about 150mM); The about 5mM of Tris HCl-is to about 100mM; PH about 6.0 is to about 9.0 (preferred about 6.5 to about 8.5); PEG (MW800-8000, preferred 3350,4000,6000 or 8000)-0 to about 25%; Protamine, the hGH of preferred 3: 1 ratios: protamine; And poly arginine, the hGH of preferred 5: 1 ratios: poly arginine.Such compositions can optionally comprise: sucrose-0mg/ml is to about 100mg/ml; Aminoacid (for example arginine and glycine)-0mg/ml is to about 50mg/ml; Antiseptic (antimicrobial, phenol, metacresol (matacrescol), benzyl alcohol, p-Hydroxybenzoate)-0% is to about 5% (preferred 0% to about 0.9%); And Polysorbate-0mg/ml is to about 10mg/ml.According to an embodiment, be feature with 80% payload according to compositions of the present invention.

Comprise about 100mM sodium acetate, about 5%PEG6000MW and about 25mM Tris HCl, pH7.5 according to the preferred preparations carrier of the present invention.Use the hGH compositions of such preparing carriers to comprise: about 9.35mg/ml crystallization hGH and about 1.81mg/ml poly arginine (or about 3.12mg/ml protamine).As skilled in the art will appreciate, suppose according to compositions of the present invention and can comprise about 1mg/ml to about 100mg/ml hGH concentration, therefore should adjust the concentration of poly arginine (or protamine), making is enough to keep 5: 1 hGH: poly arginine (w/w) ratio or 3: 1hGH: protamine (w/w) ratio and the release that keeps the hGH of low solubility and about 5ng/ml.For example, for above-described preparation, if the hGH concentration of expectation is about 20mg/ml, poly arginine (or protamine) concentration should be about 4mg/ml so.

The present invention further provides the crystalline method of preparation hGH or hGH derivant.A kind of such method comprises the steps: that (a) mixes the solution of human growth hormone or human growth hormone's derivant with crystallization solution, and described crystallization solution comprises salt and Ionomer; And (b) under the temperature between about 4 ℃ and about 37 ℃, cultivate described solution greater than about 12 hours, till the crystal that forms human growth hormone or human growth hormone's derivant.In another embodiment, described method comprises the steps: that (a) mixes the solution of human growth hormone or human growth hormone's derivant with crystallization solution, and described crystallization solution comprises salt and precipitant; And (b) under the temperature between about 4 ℃ and about 37 ℃, cultivate described solution greater than about 16 hours, till the crystal that forms human growth hormone or human growth hormone's derivant.In another embodiment, the solution in the step of above-mentioned any method (b) can be cultivated greater than an about week under about 15 ℃ temperature.In preferred embodiments, the crystal of hGH or hGH derivant is that calcium crystal, monovalent cation crystal, protamine crystal or poly arginine crystal and Ionomer are protamine or poly arginine.In another embodiment, Ionomer is polylysine or poly ornithine (polyorthinine).In another embodiment also, Ionomer is any two or more the mixture in protamine, poly arginine and the polylysine.

Salt in the step (a) of said method can be univalent also can be bivalence and can be inorganic or organic.The embodiment of preferred divalent salts is a calcium salt.In a more preferred embodiment, calcium salt is selected from the group of being made up of calcium acetate, calcium chloride, calcium gluconate and calcium sulfate.In preferred embodiment also, calcium salt is a calcium acetate.In another preferred embodiment, monovalent cation is selected from the group of being made up of lithium, sodium, potassium and ammonium.In a more preferred embodiment, monovalent cation is a sodium.

In the alternative preferred embodiment of the present invention, monovalent cation salt is sodium salt.In a more preferred embodiment, sodium salt is selected from the group of being made up of sodium citrate, sodium phosphate and sodium acetate.In preferred embodiment also, sodium salt is a sodium acetate.

In said method, when described salt was calcium salt or monovalent cation salt, it was present in the crystallization solution of step (a) with the concentration between about 0.01mM and the about 1M.In preferred embodiments, this concentration about 25 and about 205mM between.When described salt was calcium salt, it was present in the crystallization solution of step (a) with the concentration between about 0.01mM and the 235mM.

In preferred embodiments, the crystallization solution of step (a) further comprises the pH buffer.In a more preferred embodiment, buffer has the pH between about pH6 and the about pH10.In a more preferred embodiment, the pH of buffer is between about pH7.5 and about pH10.In a more preferred embodiment, the pH of buffer is between about pH7.0 and about pH10.In preferred embodiment also, the pH of buffer is between about pH6 and about pH9.In going back a preferred embodiment, the pH of buffer is between about pH7.8 and about pH8.9.

At said method on the other hand, the buffer in the step (a) is selected from the group of being made up of Tris, HEPES, acetate, phosphate, citrate, borate, imidazoles and glycine.In the preferred embodiment of said method, buffer in the step (a) is selected from by bicarbonate, imidazoles-malate, glycine, 2, two (hydroxymethyl)-2 of 2-, 2 '; 2 "-nitrilotriethanol (" two-tris "), carbonate, N-(acetylamino)-iminodiacetic acid, 2-amino-2-methyl-1,3 propylene glycol and the (group that N-(1-acetylamino)-2-aminoethane sulphonic acid is formed.

In one of said method, the precipitant that is used to prepare hGH or hGH derivative crystal is polymeric typically, comprises low-molecular-weight polyalcohols and protamine.In another embodiment of the invention, the precipitant in one of said method step (a) is a non-ionic polyalcohol.In preferred embodiments, non-ionic polyalcohol is selected from the group of being made up of alcohol, Polyethylene Glycol (PEG) and polyvinyl alcohol or ethanol.In a more preferred embodiment, precipitant is isopropyl alcohol or ethanol.In the embodiment that also is more preferably selected, PEG has the molecular weight between about 200 and about 8000.PEG is to have 3350,4000,6000 or 8000 molecular weight.In an embodiment of more preferably selecting, PEG has about 6000 molecular weight.In another embodiment preferred also, PEG with about 0.5% and about 12%w/v between concentration exist.

In another embodiment of one of said method, the precipitant in the step (a) is a non-ionic polyalcohol.In preferred embodiments, non-ionic polyalcohol is selected from the group of being made up of protamine, poly arginine, poly ornithine and polylysine.

The blend step of said method (a) comprises that the solution with hGH or hGH derivant mixes with crystallization solution.In an embodiment of described method, hGH that obtains or the concentration of hGH derivant in described crystallization solution are at about 1mg/ml and about 1, between the 000mg/ml.In preferred embodiments, hGH in described solution or hGH derivant exist with the concentration between about 2mg/ml and the about 50mg/ml.In further embodiment, hGH in described solution or hGH derivant exist with the concentration between about 10mg/ml and the about 25mg/ml.

In the preferred embodiment of the above-mentioned method for preparing hGH or hGH derivative crystal, in step (b), under about 33 ℃ temperature, cultivate the solution that comprises hGH or hGH derivant and crystallization solution and reach about 0.25 day to about two days.Alternately, described temperature can be about 37 ℃.In another embodiment, under about 25 ℃ temperature, cultivate the solution that comprises hGH or hGH derivant and crystallization solution and reach about 0.25 day to about two days.In going back another embodiment, under about 15 ℃ temperature, cultivate the solution that comprises hGH or hGH derivant and crystallization solution and reach about 0.25 day to about two days.

The present invention further provides the alternative method for preparing the crystal of hGH crystal, hGH derivant or comprise the compositions of this crystalloid and excipient.This method comprises the steps: that (a) mixes the solution of hGH or hGH derivant to produce crystallization solution with the crystallization buffer; (b) in described crystallization solution, add deionized water; (c) in described crystallization solution, add ion-type micromolecule or Ionomer; (d) in described crystallization solution, add salt; And (e) under about 10 ℃ and about 40 ℃ temperature, cultivate described crystallization solution about 2 to about 168 hours, till the crystal that forms hGH or hGH derivant.In further embodiment of the present invention, described cultivation was carried out about 4 to about 48 hours.In another preferred embodiment, at the crystallization solution of the step (e) of said method, under the temperature between about 4 ℃ and about 40 ℃, cultivated about 4 to about 48 hours, till the crystal that forms hGH or hGH derivant.According to alternate embodiment, carry out said method, after step (b), have step arbitrarily.Described step arbitrarily comprises in described crystallization solution adds precipitant.In the further embodiment of said method, step (c) is arbitrarily.No matter whether use those steps arbitrarily, in preferred embodiments, the crystallization solution about 1 of incubation step (e) was to about 2 days under the temperature between about 15 ℃ and about 37 ℃.In another preferred embodiment, under the temperature between about 4 ℃ and about 37 ℃ the crystallization solution about 1 of incubation step (e) to about 2 days.

In according to the preferred embodiments of the invention, in the step (d) of said method, described salt is calcium salt or monovalent cation salt.In preferred calcium crystal embodiment, calcium salt is selected from the group of being made up of calcium acetate, calcium chloride, calcium gluconate and calcium sulfate.In going back a preferred embodiment, calcium salt is a calcium acetate.In preferred embodiments, calcium acetate is the aqueous solution form with the pH between about 3 and about 9.0.In a more preferred embodiment, the calcium acetate aqueous solution has the pH between about 7.0 and about 8.6.In another embodiment, the calcium acetate in step (e) solution exists with the concentration between about 0.1mM and the about 205mM.In a more preferred embodiment, the calcium acetate in step (e) crystallization solution exists with the concentration between about 85mM and the about 100mM.

In a more preferred embodiment, monovalent cation salt is selected from the group of being made up of lithium, sodium, potassium and ammonium.In going back a preferred embodiment, the monovalent cation salt in the said method step (d) is sodium.Similarly, in a more preferred embodiment, monovalent cation salt is selected from the group of being made up of sodium citrate, sodium phosphate and sodium acetate.In going back a preferred embodiment, monovalent cation salt is sodium acetate.In preferred embodiments, sodium acetate is the aqueous solution form with the pH between about 3 and about 9.0.In a more preferred embodiment, aqueous sodium acetate solution has the pH between about 7.0 and about 8.6.In another embodiment, the sodium acetate in step (e) crystallization solution exists with the concentration between about 0.5mM and the about 800mM.In a more preferred embodiment, the sodium acetate in step (e) crystallization solution exists with the concentration between about 100mM and the about 500mM.Described concentration also can be between about 85mM and about 100mM.

In going back another embodiment preferred, hGH or hGH derivant in said method step (e) crystallization solution exist with the concentration between about 2mg/ml and the about 17.5mg/ml.In another preferred embodiment, hGH in said method step (e) crystallization solution or hGH derivant exist with the concentration between about 14.5mg/ml and the about 15.5mg/ml.In further embodiment, hGH or hGH derivant in step (e) crystallization solution exist with the concentration between about 2mg/ml and the about 100mg/ml.

In preferred embodiments, the crystallization buffer in said method step (a) is selected from the group of being made up of Tris-HCl, HEPES, acetate, phosphate, citrate, borate, imidazoles and glycine.Alternately; the crystallization buffer is selected from by Tris-HCl, glycine, HEPES, imidazoles, two-Tris, AMP (2-amino-2 methylpropanol), AMPD (2-amino-2-methyl-1; ammediol), AMPSO (3-([1,1-dimethyl-2-hydroxyethyl] amino)-2-hydroxypropanesulfonic acid), N-two (ethoxy) glycine, ethanolamine, glyclglycine, TAPS, taurine (Taurin)

, the group formed of Triane and their mixture.In another preferred embodiment, the crystallization buffer in step (a) solution exists with the concentration between about 10mM and the about 800mM.

In another embodiment of said method, the crystallization buffer in the step (a) exists with the pH between about 3 and about 10.In preferred embodiments, the crystallization buffer exists with the pH between about 6 and about 9.In going back another embodiment preferred, the crystallization buffer exists with the pH between about 7.5 and about 10.

In another preferred embodiment, the pH of the crystallization buffer in said method step (e) solution is between about 3 and about 10.In a more preferred embodiment, the pH of the crystallization buffer in the solution is between about 6 and about 9.5.In going back a preferred embodiment, the pH of the crystallization buffer in the solution is between about 7.5 and about 9.5.

In the preferred embodiment of the method for the arbitrary steps after those comprise the step (b) of adding precipitant in crystallization solution, described precipitant is nonionic micromolecule or non-ionic polyalcohol.In preferred embodiments, non-ionic polyalcohol is selected from the group of being made up of Polyethylene Glycol (PEG), polyvinyl alcohol and their mixture.In another preferred embodiment, PEG have be selected from by between about 200 and about 8000, about 6000, about 4000 and the molecular weight of about 3350 groups of forming.In another embodiment preferred also, PEG with about 0.5% and about 20% (w/v) between concentration be present in the crystallization solution.In another preferred embodiment of said method, precipitant is selected from the group of being made up of aminoacid, peptide, polyamino acid and their mixture.

In another preferred embodiment, in the step (c) of said method, Ionomer is selected from the group of being made up of protamine, poly arginine, polylysine, poly ornithine and eight arginine (octarginine).In another preferred embodiment, in the step (c) of said method, with the hGH between 5: 1 and about 1: 25: the ratio of poly arginine adds poly arginine.Under the temperature between about 15 ℃ and about 37 ℃, cultivate the crystallization solution that obtains and reach about 16 to about 48 hours.

The present invention also provide the preparation human growth hormone crystal, human growth hormone's derivant crystal or comprise the also another kind of method of the compositions of this crystalloid and excipient.This method comprises the steps: that (a) mixes the solution of human growth hormone or human growth hormone's derivant to produce crystallization solution with the crystallization buffer; (b) in described crystallization solution, add deionized water; (c) in described crystallization solution, add precipitant; (d) in described crystallization solution, add salt; (e) under the temperature between about 10 ℃ and about 40 ℃, cultivate described crystallization solution about 2 to about 168 hours, till the crystal that forms human growth hormone or human growth hormone's derivant, and (f) in the crystal of described human growth hormone or human growth hormone's derivant, add Ionomer.An embodiment according to the method described above, step (f) is arbitrarily.In preferred embodiments, under the temperature between about 15 ℃ and about 37 ℃, the crystallization solution of incubation step (e) reaches about one to about two days.In another preferred embodiment, under the temperature between about 4 ℃ and about 37 ℃, the crystallization solution of incubation step (e) reaches about one to about two days.In further embodiment of the present invention, described cultivation was carried out about 2 to about 48 hours.In another preferred embodiment, under the temperature between about 4 ℃ and about 40 ℃, the crystallization solution of cultivating said method step (e) reaches about 4 to about 48 hours, till the crystal that forms hGH or hGH derivant.

In according to the preferred embodiments of the invention, in the step (d) of said method, described salt is calcium salt or monovalent cation salt.In a more preferred embodiment, described calcium salt is selected from the group of being made up of calcium acetate, calcium chloride, calcium gluconate and calcium sulfate.In going back a preferred embodiment, described calcium salt is a calcium acetate.In preferred embodiments, calcium acetate is the aqueous solution form with the pH between about 3 and about 9.0.In a more preferred embodiment, the calcium acetate aqueous solution has the pH between about 7.0 and about 8.6.In another embodiment, the calcium acetate in step (e) crystallization solution exists with the concentration between about 0.1mM and the about 205mM.In a more preferred embodiment, the calcium acetate in step (e) crystallization solution exists with the concentration between about 85mM and the about 100mM.

In a more preferred embodiment, monovalent cation is selected from the group of being made up of lithium, sodium, potassium and ammonium.In going back a preferred embodiment, monovalent cation is a sodium.Similarly, in a more preferred embodiment, monovalent cation salt is selected from by sodium citrate, sodium phosphate and closes the group that sodium acetate is formed.In going back a preferred embodiment, monovalent cation salt is sodium acetate.In preferred embodiments, sodium acetate is the aqueous solution form with the pH between about 3 and about 9.0.In a more preferred embodiment, aqueous sodium acetate solution has the pH between about 7.0 and about 8.6.In another embodiment, the sodium acetate in step (e) solution exists with the concentration between about 0.5mM and the about 800mM.In a more preferred embodiment, the calcium acetate in step (e) crystallization solution exists with the concentration between about 100mM and the about 500mM.Alternately, described concentration can also be between about 85mM and about 100mM.

In going back another embodiment, hGH or hGH derivant in step (e) crystallization solution of said method exist with the concentration between about 2mg/ml and the about 17.5mg/ml.In another preferred embodiment, hGH in step (e) crystallization solution or hGH derivant exist with the concentration between about 14.5mg/ml and the about 15.5mg/ml.In further embodiment, hGH or hGH derivant in step (e) crystallization solution of said method exist with the concentration between about 2mg/ml and the about 100mg/ml.

In preferred embodiments, the crystallization buffer of the step of said method (a) is selected from the group of being made up of Tris-HCl, HEPES, acetate, phosphate, citrate, borate, imidazoles and glycine.In another preferred embodiment, the crystallization buffer in step (a) solution exists with the concentration between about 10mM and the about 800mM.

In another preferred embodiment, in said method step (e) solution pH of crystallization buffer between about 3 and about 10.In a more preferred embodiment, in the solution pH of crystallization buffer between about 6 and about 9.5.In going back a preferred embodiment, the pH of crystallization buffer is between about 7.5 and about 9.5 in the solution.

In preferred embodiments, in said method step (c), precipitant is nonionic micromolecule or non-ionic polyalcohol.In preferred embodiments, non-ionic polyalcohol is selected from the group of being made up of Polyethylene Glycol (PEG), polyvinyl alcohol and their mixture.In another preferred embodiment, PEG have be selected from by between about 200 and about 8000, about 6000, about 4000 and the molecular weight of about 3350 groups of forming.In another embodiment preferred also, PEG with about 0.5% and the concentration of about 20% (w/v) be present in the crystallization solution.In another preferred embodiment, in said method step (c), precipitant is selected from the group of being made up of aminoacid, peptide, polyamino acid and their mixture.

In another preferred embodiment, in said method step (f), Ionomer is selected from the group of being made up of protamine, poly arginine, polylysine, poly ornithine and eight arginine.In another preferred embodiment, in said method step (f), with the hGH between about 5: 1 and about 1: 25: (mg: mg) ratio adds poly arginine to poly arginine.In an alternative embodiment, with the hGH between about 1: 5 and about 1: 25: (mg: mg) ratio adds poly arginine to poly arginine.The solution of cultivating in the step (f) that obtains under the temperature between about 15 ℃ and about 37 ℃ reaches about 16 to about 48 hours.By dissolving of the influence of required washing times reflection polymer fully to the dissolution of crystals speed of hGH or hGH derivant.For dissolving fully, the contrast crystal needs about 7 to about 13 washings, and for dissolving fully, needs the identical washing of the dissolving buffer between about 30 to 90 times with the crystal of poly arginine preparation.Washing is the washing step (seeing embodiment 5) in the continuous-dissolution process.

In said method in any alternate embodiment, calcium salt or monovalent cation salt can with between about 0.01M and the about 1M or the concentration between about 25mM and the about 205mM be present in the crystallization solution.In any alternate embodiment, cultivate crystallization solution in said method, time and temperature are selected from by under about 33 ℃ temperature, about 0.25 day to about two days; Under about 25 ℃ temperature, about 0.25 day to about two days and under about 15 ℃ temperature, the group of forming in about 0.25 day to about two days.

The present invention comprises that also screening is used for the treatment of the hGH in the preparation or the crystalline method of hGH derivant.The step of these class methods comprises: (1) washs described hGH or hGH derivant with the dissolving buffer under 37 ℃ temperature crystal (for example, the dissolving buffer of the crystal of 2mg and 1ml) and (2) be determined in the described dissolving buffer the described hGH of each washing or the crystalline dissolution in vitro speed of hGH derivant, wherein said crystalline described dissolution in vitro speed is between about 10 minutes and about 1500 minutes, described crystal between about 2% and about 16%, the each washing step in the continuous-dissolution process about 2 minutes to about 32 minutes (seeing embodiment 5).Described crystalline dissolution in vitro speed can also be in about 15 minutes the described crystal between about 4% and about 10% or in about 15 minutes about 0.04 and about 0.32mg between described crystal.

In order to understand the present invention better, the statement the following examples.These embodiment should not be interpreted as limiting the scope of the invention in any manner only for purposes of illustration.

Embodiment

Used following material among Chen Shu the embodiment below.

Material

Commercially available recombinant human somatropin (rhGH) is from BresaGen Ltd. (Thebarton, Australia), Polyethylene Glycol with 4000 or 6000 (PEG-4000 or PEG-6000) mean molecule quantity is from Hampton Research (Laguna Niguel, California) and protamine sulfate available from Fisher from ICN BiomedicalsInc. (Pittsburgh, PA).(Pittsburgh PA) obtains from Fisher separately for ammonium phosphate, Tris-HCl, sodium citrate, disodium hydrogen phosphate, calcium acetate, calcium chloride, zinc acetate, HEPES, sodium chloride, potassium chloride, Hydrazoic acid,sodium salt, isopropyl alcohol (IPA), ethanol and Polyethylene Glycol monomethyl ether.(Worcester, MA) or from Biomedical Research Labora tories, (Worcester MA) obtains Inc. the Sprague-Dawley rat from Charles River Laboratories.Poly arginine obtains from Sigma (St.Louis).

Analytical technology and test

Reversed phase high-performance liquid chromatography.Be equipped with C5,5cm * 4.6mm, ((Palo Alto CA) goes up acquisition reversed-phase high-performance liquid chromatography (RP-HPLC) to Agilent 1100 serial HPLC PA) to 3 μ m posts for Supelco, Bellefonte.Sample dissolution is being dissolved buffer (50mM HEPES pH7.2,140mM NaCl, 10mMKCl and 0.02% (v/v) NaN ₃) in and before injection, filter (0.2 μ m).The gradient method that uses solvent orange 2 A and B is 214 and 280nm monitoring elution curve.Solvent orange 2 A is made up of 99.9% deionized water/0.1%TFA.Solvent B is made up of 99.9% acetonitrile/0.1%TFA.All chemical substances all are the HPLC levels, obtain from Fisher.Use 0-2 minute 40-50%B, the gradient of 2-12 minute 50-60%B and 12-1560-85%B was carried out eluting 15 minutes.Keep the flow velocity of 1ml/min and 35 ℃ column temperature by operation.Use Agilent chemical work station software (Palo Alto, CA) analytical data.

Use solvent orange 2 A (99.9% deionized water/0.1%TFA) and B (protamine in the gradient method working sample preparation of 99.9% acetonitrile/0.1%TFA) or the concentration of poly arginine.Use 0-2.5 minute 95: 5 (A: B), 2.5-7.5 minutes 65: 35 (A: B), 7.5-15.5 minute 25: 75 (A: B), 15.5-17.0 minute 25: 75 (A: B), 17-17.1 minutes 95: 5 (A: gradient design B), carried out eluting 20 minutes with the flow velocity of 1ml/min.Obtained the eluting of typical protamine or poly arginine at 6.2 minutes, go out complete hGH at 14 minutes eluting.Measuring AUC at 213nm calculates.Calculate protamine and/or poly arginine content of additive (mg/ml) by standard curve, described standard curve is by each generation of each additive.Can use this identical method to analyze the excipient that from complex, discharges.

With separately but similar inversion method is measured the hGH of relevant degraded.For example, on C5Supelco Discovery Bio Wide Pore post (5cm * 4.6mm, 3 μ m particle diameters, 30nm aperture), analyze, in whole service, keep 37 ℃ thermostat temperature.Use has mobile phase A (20%ACN, 80%H ₂O, 0.1%TFA) and Mobile phase B (20%ACN, 80%2-propanol, 01.%TFA) gradient method monitoring elution curve.Gradient system was changed to 45%B from 20% in 0 to 5 minute, in 5 to 15 minutes, be changed to 55%B from 45%, be changed to 90%B from 55% in 15 to 15.1 minutes, 90%B is fixing up to 17 minutes and after this step, rebuilds upright 20%B until reaching 20 minutes.

The size exclusion chromatography.Be equipped with TSK-Gel G2000SWXL post (part#; 08450, Tosoh Biosep LLC, Montgomeryville, PA) (Palo Alto CA) goes up acquisition efficient size exclusion chromatograph (SEC-HPLC) to the Agilent 1100 serial HPLC of (7.8mm * 30cm, 5 μ m) and Agilent 1100 serial MWD (UV).Sample dissolution is filtered with 0.2 μ m in 0.2ml dissolving buffer and before injecting Agilent 1100 series of temperatures control autopipette.214 and 280nm monitoring elution curve, mobile phase is 50mM Tris-HCl, 150mM Nacl, 0.05%NaN ₃, pH7.5.Keep column temperature at 25 ℃, use Agilent 1100 serial degassers that solvent is outgased.

UV-VIS absorbs and optical microscopy.At Beckman Coulter Inc., Fullerton obtains the UV-VIS spectrophotometric spectra on Beckman DU 740 spectrophotometers of CA.By using Olympus BX-51 microscopical bright field imaging and obtaining amplifying light micrograph under the 40x to 400x with Sony DXC-970MD 3CCD colorful digital video camera, use Media Cybernetics L.P., Silver Springs, the Image-Pro software of Maryland.

Transmission electron microscopy (TEM).Tem analysis carries out as follows.Twice of the hGH crystal suspension that washes with water in the mother solution used 0.5% uranyl acetate negative staining 1 hour then to remove excessive mother solution.(1-5 μ L) transfers on the copper TEM grid with painted hGH crystal suspension.Excessive liquid pours off (wicked away) and air drying sample grid momently.The TEM grid is transferred on the sample stage of JEOL 1210 transmission electron microscopes and used the 80KV electron beam to collect image.In crystal, observe and organize extraordinary lattice structure, have and the prismatic axle of crystal orientation in line.

Embodiment 1

With ammonium phosphate crystallization hGH.At first commercially available hGH (50mg) is dissolved in 15ml Tris-HCl (10mM, pH8.0) also use in and have 10, (10mM's 000 molecular weight cut value (cut off) Pierce dialysis apparatus tube (MWCO) pH8.0) dialyses facing to 2 * 4000ml Tris-HCl.Regulated protein concentration at the centrifugal 20-30 of 4000rpm minute by using Millipore concentrator (MWCO10,000).As at 280nm/0.813 (1mg/ml hGH A ₂₈₀=0.813 absorbance units) by absorptiometry, the concentration of finding hGH is in the scope of 30-45mg/ml.In solution, add deionized water to produce the whole protein concentration of 10-20mg/ml.By in solution, adding ammonium phosphate (NH ₄H ₂PO ₄) (2.5M; PH8.9) consequently obtain 860mM NH ₄H ₂PO ₄Final concentration, generate the crystal of hGH.Cultivated solution 16 hours down at 25 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.Find that the crystal length that obtains is about 8 to 15 μ m, crystallization yield is greater than 90%.See Fig. 1.

Embodiment 2

With sodium citrate crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 17.5mg/ml.By in solution, adding sodium citrate (1.5M), consequently obtain the final concentration of 390mM sodium citrate, generate the crystal of hGH.Do not need pH regulator, except that the hGH in 10mM Tris-HCl.Cultivated solution 16 hours down at 25 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.The crystal length of finding to obtain is less than 8 μ m, and crystallization yield is greater than 85%.See Fig. 2.

Embodiment 3

With sodium phosphate crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 12.5-17.5mg/ml.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.By in solution, adding sodium hydrogen phosphate (Na ₂HPO ₄) (1M) generate the crystal of hGH, so that obtain 600mM Na ₂HPO ₄Final concentration.Cultivated solution 16 hours down at 25 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.The crystal length of finding to obtain is between 5 and 25 μ m, and crystallization yield is greater than 75%.See Fig. 3.

Embodiment 4

With calcium acetate and protamine sulfate crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 15mg/ml.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.In solution, add the final concentration of protamine sulfate to 1mg/ml.By in solution, adding calcium acetate (1M), consequently obtain the final concentration of 85mM calcium acetate, generate the crystal of hGH.Cultivated solution 8 hours down at 37 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.The crystal length of finding to obtain is less than 20 μ m, and crystallization yield is greater than 70%.See Fig. 4.

Embodiment 5

The crystalline solubility characteristics of hGH by the preparation of salt induced crystallization.After cultivating the crystallization solution among the embodiment 1-4, the precipitation crystal is also removed remaining supernatant.Move or vortex is resuspended in 0.200ml dissolving buffer (50mM HEPES (pH7.2), 140mM NaCl, 10mM KCl and 0.02% (v/v) NaN with crystal settling piece (0.4mg) by suction ₃) in, then about 15 minutes of 37 ℃ of balances.Then 10, about 2 minutes of the centrifugal sample of 000xg and remove supernatant fully and be used for the determination of protein concentration measured at 280nm by RP-HPLC, SEC-HPLC or UV-VIS.Further the crystal settling piece is resuspended in the 0.200ml dissolving buffer and repeats said process up in supernatant, measuring less than detectable protein.This process is called as continuous-dissolution.

Fig. 5 has shown in the foregoing description 1-4 with monovalence (Na or NH ₄) or the crystalline conduct of different hGH of bivalence (Ca) salt preparation minute being the dissolubility behavior of the time function of unit.The hGH dissolving is discharged mapping with the accumulative perception derived from RP-HPLC, wherein uses the UV-VIS spectrophotometer to measure the AUC value of protein example with mg/ml.Data declaration is by hGH crystal dissolving fully after 60 minutes of adding the preparation of 390mM sodium citrate.In addition, by adding 600mM Na ₂HPO ₄Or 860mM NH ₄H ₂PO ₄The dissolving fully after 60 or 75 minutes respectively of the hGH crystal of preparation.On the other hand, the hGH crystal dissolving (seeing the following form 1) fully after 390 minutes by adding the preparation of 85mM calcium acetate and protamine sulfate.

Table 1. is tested at the continuous-dissolution of hGH salt in the dissolving buffer that 280nm measures

-protein concentration is expressed as the percentage ratio that release amounts to

Time (branch)	390mM sodium citrate (embodiment 2)	600mM Na ₂HPO ₄(embodiment 3)	860mM NH ₄H ₂PO ₄(embodiment 1)	85mM calcium acetate+protamine (embodiment 4)
Time (branch)	390mM sodium citrate (embodiment 2)	600mM Na ₂HPO ₄(embodiment 3)	860mM NH ₄H ₂PO ₄(embodiment 1)	85mM calcium acetate+protamine (embodiment 4)	0	0.00	0.00	0.00	0.00
15	71.59	78.99	93.77	8.53	0	0.00	0.00	0.00	0.00
15	71.59	78.99	93.77	8.53	30	99.36	99.85	99.18	19.39
45	99.99	99.99	99.50	26.81	30	99.36	99.85	99.18	19.39
45	99.99	99.99	99.50	26.81	60	100.00	100.00	99.50	34.92
75	100.00	100.00	100.00	38.31	60	100.00	100.00	99.50	34.92
75	100.00	100.00	100.00	38.31	90	100.00	100.00	100.00	42.22

105	100.00	100.00	100.00	46.26
105	100.00	100.00	100.00	46.26	120	100.00	100.00	100.00	49.62
135	100.00	100.00	100.00	52.73	120	100.00	100.00	100.00	49.62
135	100.00	100.00	100.00	52.73	150	100.00	100.00	100.00	55.08
165	100.00	100.00	100.00	57.20	150	100.00	100.00	100.00	55.08
165	100.00	100.00	100.00	57.20	180	100.00	100.00	100.00	59.65
195	100.00	100.00	100.00	63.95	180	100.00	100.00	100.00	59.65
195	100.00	100.00	100.00	63.95	210	100.00	100.00	100.00	67.57
225	100.00	100.00	100.00	69.17	210	100.00	100.00	100.00	67.57
225	100.00	100.00	100.00	69.17	240	100.00	100.00	100.00	71.63
255	100.00	100.00	100.00	74.35	240	100.00	100.00	100.00	71.63
255	100.00	100.00	100.00	74.35	270	100.00	100.00	100.00	76.85
285	100.00	100.00	100.00	78.39	270	100.00	100.00	100.00	76.85
285	100.00	100.00	100.00	78.39	300	100.00	100.00	100.00	81.06
315	100.00	100.00	100.00	83.97	300	100.00	100.00	100.00	81.06
315	100.00	100.00	100.00	83.97	330	100.00	100.00	100.00	87.97
345	100.00	100.00	100.00	90.57	330	100.00	100.00	100.00	87.97
345	100.00	100.00	100.00	90.57	360	100.00	100.00	100.00	94.20
375	100.00	100.00	100.00	98.28	360	100.00	100.00	100.00	94.20
375	100.00	100.00	100.00	98.28	390	100.00	100.00	100.00	100.00

Embodiment 6

With calcium acetate and 10% isopropyl alcohol crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 15mg/ml.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.By in solution, adding calcium acetate (1M), consequently obtain the final concentration of 85mM calcium acetate, generate the crystal of hGH.In this solution, add 10% (v/v) isopropyl alcohol (IPA).Cultivated solution 16 hours down at 25 ℃ then.Obtain rhabdolith and pass through the optical microscopy imaging.The crystal length that find to obtain is greater than 100 μ m, and crystallization yield is greater than 85%.See Fig. 6.

Embodiment 7

With calcium chloride and 5% isopropyl alcohol crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 15mg/ml.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.By in solution, adding calcium chloride (CaCl ₂) (1M), consequently obtain the final concentration of 85mM calcium chloride, generate the crystal of hGH.In this solution, add 5% (v/v) isopropyl alcohol (IPA).Cultivated solution 16 hours down at 25 ℃ then.Obtain rhabdolith and pass through the optical microscopy imaging.The crystal length that find to obtain is greater than 200 μ m, and crystallization yield is greater than 85%.See Fig. 7.

Embodiment 8

With 10%PEG-6000 and 10% alcohol crystal hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 25mg/ml.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.By in solution, adding the crystal that 10% (v/v) PEG-6000 and 10% (v/v) ethanol (EtOH) generate hGH.Cultivated solution 16 hours down at 37 ℃ then.Obtain rhabdolith and pass through the optical microscopy imaging.The crystal length of finding to obtain is less than 25 μ m, and crystallization yield is greater than 70%.See Fig. 8.

Embodiment 9

The crystalline solubility characteristics of hGH with the alcohol preparation.After cultivating the crystallization solution for preparing among the embodiment 6-8, the precipitation crystal is also removed remaining supernatant.Move or vortex is resuspended in the crystal settling piece in the 0.200ml dissolving buffer (seeing embodiment 5) by suction, then about 15 minutes of 37 ℃ of balances.Then 10, the centrifugal sample of 000xg 2 minutes and remove supernatant and be used for the determination of protein concentration measured at 280nm by RP-HPLC, SEC-HPLC or UV-VIS.HGH dissolving is measured as accumulative perception and derived from AUC value or UV-VISmg/ml measured value.Further the crystal settling piece is resuspended in the dissolving buffer and repeats said process up in supernatant, measuring less than detectable protein.

Fig. 9 and table 2 have illustrated with the hGH crystal of 10%IPA/85mM calcium acetate, 5%IPA/85mM calcium chloride and 10% ethanol/10%PEG-6000 preparation as the dissolubility behavior with the time function that is divided into unit.The result confirms, by hGH crystal dissolving fully after 60 minutes of adding the preparation of 390mM sodium citrate.In addition, by hGH crystal dissolving fully after 150 minutes of adding the preparation of 10%IPA/85mM calcium acetate, and the hGH crystal dissolving fully after 120 minutes and 135 minutes respectively by adding 5%IPA/85mM calcium chloride and 10% ethanol/10%PEG-6000 preparation.

The table 2. crystalline dissolution in vitro result of hGH (embodiment 6-8) of ethanol preparation -protein concentration measures and is represented as the percentage ratio that discharges total amount at 280nm

0	0.00	0.00	0.00
0	0.00	0.00	0.00	15	19.39	70.16.	76.12
30	53.88	82.84	86.50	15	19.39	70.16.	76.12
30	53.88	82.84	86.50	45	74.59	92.33	92.33
60	84.13	96.03	94.62	45	74.59	92.33	92.33
60	84.13	96.03	94.62	75	90.64	98.48	94.87
90	95.47	99.91	96.28	75	90.64	98.48	94.87
90	95.47	99.91	96.28	105	98.32	99.94	96.82
120	99.51	100.00	99.68	105	98.32	99.94	96.82
120	99.51	100.00	99.68	135	99.51	100.00	100.00
150	100.00	100.00	100.00	135	99.51	100.00	100.00
150	100.00	100.00	100.00	165	100.00	100.00	100.00
180	100.00	100.00	100.00	165	100.00	100.00	100.00

Embodiment 10

With calcium acetate and 2%PEG-6000 crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 15mg/ml.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.In this solution, add 2% (v/v) PEG-6000.By in solution, adding calcium acetate (1M), consequently obtain the final concentration of 85mM calcium acetate, generate the crystal of hGH.Cultivated solution 16 hours down at 25 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.The crystal length that find to obtain is about 25 and about 75 μ m, and crystallization yield is greater than 85%.See Figure 10.

Embodiment 11

With sodium acetate and 6%PEG-6000 crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 15mg/ml.(1M is pH8.6) to the final concentration of 100mM for interpolation/Tris-HCl.In this solution, add 6% (v/v) PEG-6000.By in solution, adding sodium acetate (1M), consequently obtain the final concentration of 500mM sodium acetate, generate the crystal of hGH.Cultivated solution 16 hours down at 25 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.The crystal length that find to obtain is about 25 and about 75 μ m, and crystallization yield is greater than 85%.See Figure 11.

Embodiment 12

With calcium chloride and 6%PEG-6000 crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 15mg/ml.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.In this solution, add 6% (v/v) PEG-6000.By in solution, adding CaCl ₂(1M), consequently obtain 85mM CaCl ₂Final concentration, generate the crystal of hGH.Cultivated solution 16 hours down at 25 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.The crystal length that find to obtain is between greater than 100 μ m, and crystallization yield is greater than 90%.See Figure 12.

Embodiment 13

With calcium acetate, 6%PEG-6000 and protamine sulfate crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.Add deionized water in molten night to produce the whole protein concentration of 15mg/ml to spissated hGH.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.In this solution, add protamine sulfate (1mg/ml) and 6%PEG-6000 (v/v).By in solution, adding calcium acetate (1M), consequently obtain the final concentration of 85mM calcium acetate, generate the crystal of hGH.Cultivated solution 16 hours down at 37 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.The crystal length of finding to obtain is less than 25 μ m, and crystallization yield is greater than 70%.See Figure 13.

Embodiment 14

With calcium acetate and 6%PEG-MME-5000 crystallization hGH.Described in embodiment 1, purification also concentrates commercially available hGH.In spissated hGH solution, add deionized water to produce the whole protein concentration of 15mg/ml.(1M is pH8.6) to the final concentration of 100mM to add Tris-HCl.In this solution, add Polyethylene Glycol monomethyl ether (PEG-MME-500).By in solution, adding calcium acetate (1M), consequently obtain the final concentration of 125mM calcium acetate, generate the crystal of hGH.Cultivated solution 16 hours down at 25 ℃ then.Obtain acicular crystal and pass through the optical microscopy imaging.The crystal length of finding to obtain is less than 50 μ m, and crystallization yield is greater than 90%.See Figure 14.

Embodiment 15

The crystalline solubility characteristics of hGH with the Polyethylene Glycol preparation.After cultivating the crystallization solution for preparing among the embodiment 10-14, the precipitation crystal is also removed remaining supernatant.Move or vortex is resuspended in the crystal settling piece in the 0.2ml dissolving buffer (seeing embodiment 5) by suction, then about 15 minutes of 37 ℃ of balances.Then 10, the centrifugal sample of 000xg 2 minutes and remove supernatant and be used for the determination of protein concentration measured at 280nm by RP-HPLC, SEC-HPLC or UV-VIS.Further the crystal settling piece is resuspended in the dissolving buffer and repeats said process up in supernatant, measuring less than detectable protein.

Figure 15 and table 3 have illustrated with 2%PEG-6000/85mM calcium acetate, 6%PEG-6000/500mM sodium acetate, 6%PEG-6000/85mM CaCl ₂, the preparation of 6%PEG-6000A/85mM calcium acetate/protamine and 6%PEG-MME-5000/125mM calcium acetate the hGH crystal as dissolubility behavior with the function of time of being divided into unit.HGH dissolving is measured as accumulative perception and derived from AUC value or UV-VISmg/ml measured value.The result confirms that the slowest by the hGH dissolution of crystals that adds 6%PEG-6000/85mM calcium acetate/protamine preparation, appearance is dissolved fully after 495 minutes.Crystalline other crystal of interpolation 2%PEG-6000/85mM calcium acetate dissolved in the time of 300 minutes or other hGH crystal is still less dissolving in the time.

Table 3. is tested at PEG and the continuous-dissolution of hGH salt in the dissolving buffer that 280nm measures

-protein solubility is represented as the percentage ratio that discharges total amount

Time (branch)	2% PEG-6000/85 mM calcium acetate (embodiment 10)	6%PEG-6000/ 500mM sodium acetate (embodiment 11)	6% PEG-6000/85 mM calcium chloride (embodiment 12)	6% PEG-6000/85 mM calcium acetate/protamine sulfate (embodiment 13)	6% PEG-MME-5000/ 125mM calcium acetate (embodiment 14)
Time (branch)	2% PEG-6000/85 mM calcium acetate (embodiment 10)	6%PEG-6000/ 500mM sodium acetate (embodiment 11)	6% PEG-6000/85 mM calcium chloride (embodiment 12)		6% PEG-MME-5000/ 125mM calcium acetate (embodiment 14)	0	0.00	0.00	0.00	0.00	0.00
15	8.41	14.23	6.63	5.66	9.50	0	0.00	0.00	0.00	0.00	0.00
15	8.41	14.23	6.63	5.66	9.50	30	16.80	23.03	19.50	11.58	28.46
45	27.64	34.74	37.74	17.22	48.04	30	16.80	23.03	19.50	11.58	28.46
45	27.64	34.74	37.74	17.22	48.04	60	35.57	47.34	54.60	21.25	62.61
75	48.75	65.16	67.67	24.63	73.76	60	35.57	47.34	54.60	21.25	62.61
75	48.75	65.16	67.67	24.63	73.76	90	56.18	78.86	77.90	28.15	82.70
105	62.70	88.66	85.26	31.77	91.15	90	56.18	78.86	77.90	28.15	82.70
105	62.70	88.66	85.26	31.77	91.15	120	66.49	90.36	90.59	34.05	95.70
135	70.07	90.36	95.18	38.83	98.18	120	66.49	90.36	90.59	34.05	95.70
135	70.07	90.36	95.18	38.83	98.18	150	72.87	90.36	98.04	40.60	99.60
165	74.82	90.58	100.00	43.28	100.00	150	72.87	90.36	98.04	40.60	99.60
165	74.82	90.58	100.00	43.28	100.00	180	90.23	93.06	100.00	45.69	100.00
195	90.23	95.80	100.00	47.52	100.00	180	90.23	93.06	100.00	45.69	100.00
195	90.23	95.80	100.00	47.52	100.00	210	90.23	100.00	100.00	51.27	100.00
225	92.90	100.00	100.00	53.38	100.00	210	90.23	100.00	100.00	51.27	100.00
225	92.90	100.00	100.00	53.38	100.00	240	92.90	100.00	100.00	55.31	100.00
255	96.61	100.00	100.00	57.24	100.00	240	92.90	100.00	100.00	55.31	100.00
255	96.61	100.00	100.00	57.24	100.00	270	96.61	100.00	100.00	58.61	100.00
285	96.61	100.00	100.00	60.28	100.00	270	96.61	100.00	100.00	58.61	100.00
285	96.61	100.00	100.00	60.28	100.00	300	100.00	100.00	100.00	64.90	100.00
315	100.00	100.00	100.00	68.04	100.00	300	100.00	100.00	100.00	64.90	100.00
315	100.00	100.00	100.00	68.04	100.00	330	100.00	100.00	100.00	72.46	100.00
345	100.00	100.00	100.00	76.26	100.00	330	100.00	100.00	100.00	72.46	100.00
345	100.00	100.00	100.00	76.26	100.00	360	100.00	100.00	100.00	79.36	100.00
375	100.00	100.00	100.00	83.20	100.00	360	100.00	100.00	100.00	79.36	100.00
375	100.00	100.00	100.00	83.20	100.00	390	100.00	100.00	100.00	86.17	100.00
405	100.00	100.00	100.00	89.15	100.00	390	100.00	100.00	100.00	86.17	100.00
405	100.00	100.00	100.00	89.15	100.00	420	100.00	100.00	100.00	92.25	100.00
435	100.00	100.00	100.00	94.40	100.00	420	100.00	100.00	100.00	92.25	100.00
435	100.00	100.00	100.00	94.40	100.00	450	100.00	100.00	100.00	95.96	100.00
465	100.00	100.00	100.00	98.07	100.00	450	100.00	100.00	100.00	95.96	100.00
465	100.00	100.00	100.00	98.07	100.00	480	100.00	100.00	100.00	99.07	100.00
495	100.00	100.00	100.00	100.00	100.00	480	100.00	100.00	100.00	99.07	100.00

Embodiment 16

Use the pharmacokinetic study of Sprague-Dawley rat.Subcutaneous soluble (commercially available) or the crystal that gives 24 female Sprague-Dawley rat 2.5mg/kg dosage (hGH (pressing the preparation described in the embodiment 10) suspension of 85mM calcium acetate/2%PEG-6000).The average weight of every rat is 200 grams.Described 24 rats are divided into two groups.Every group of subsystem that comprises 3 groups that contain 4 rats.In each subsystem, collect hemorrhage via jugular vein conduit implant with three specific time points.Because the amount of the blood that point can extract is limited in preset time, use leapfrog (leap frog) design.In order to keep the stability of animal, the animal subsystem in the group is hemorrhage at the time point of note.Edit blood serum sample then to form the timeline (timeline) of linear progression.By put the variation bioassay standard deviation of the meansigma methods of serum levels and described subsystem in the subsystem in preset time.See Table 4-6.In table 4-5, specified animal 1-12 accepts soluble hGH and animal 13-24 accepts crystalline hGH, and the dosage of every animal is 500 μ g.

Figure 16 has illustrated the level as hGH in the serum of the function of time of soluble and crystalline hGH.The half-life of crystallization hGH is almost high 19 times than the half-life of solvable hGH.Concerning crystalline hGH, the time that occurs maximum hGH in the serum is 4 hours, and is 0.5 hour to soluble hGH.Suppose concentration with 5.5mg/ml, equal the dosage of 2.2mg/kg, handle group and the subsystem of rat with soluble hGH or crystalline hGH, following listed cmax value shows in the table 6, compare with identical solvable dosage, the hGH that sends with crystal form significantly reduces maximum serum-concentration.In addition, soluble hGH is similar to the AUC of the crystalline total serum level of hGH, and this shows not appreciable impact of crystallization bioavailability.Calculated solvable and the T crystallization result _90%Value.The time that total AUC of this parameter indicating 90% takes place.Higher T _90%Value shows that medicine is retained in the longer time in the serum.Contained T in the table 6 _90%The result clearly illustrates that the hGH level that crystal form causes raising is than the remarkable longer time of soluble form.

The hGH animal pharmaceuticals dynamics research result of the solvable hGH of table 4.

Animal	The blood-letting time (hour)	Average hGH (ng/ml) in the serum	Standard deviation
Animal	The blood-letting time (hour)	Average hGH (ng/ml) in the serum	Standard deviation	1-4	0	0.00	0.00
5-8	0.5	1171.65	116.03	1-4	0	0.00	0.00
5-8	0.5	1171.65	116.03	9-12	1	924.49	67.90
1-4	2	726.84	163.83	9-12	1	924.49	67.90
1-4	2	726.84	163.83	5-8	4	205.90	29.40
9-12	6	17.48	6.66	5-8	4	205.90	29.40
9-12	6	17.48	6.66	1-4	8	1.14	1.68
5-8	12	0.00	0.00	1-4	8	1.14	1.68
5-8	12	0.00	0.00	9-12	24	0.00	0.00
	Amount to=	3047.50		9-12	24	0.00	0.00

Table 5. crystallization hGH (the 85mM calcium acetate/2%PEG-6000)

HGH animal pharmaceuticals dynamics research result

Animal	Time (hour)	Average hGH (ng/ml) in the serum	Standard deviation
Animal	Time (hour)	Average hGH (ng/ml) in the serum	Standard deviation	13-16	0	0.00	0.00
17-20	0.5	151.39	60.30	13-16	0	0.00	0.00
17-20	0.5	151.39	60.30	21-24	1	159.19	69.50
13-16	2	236.64	75.70	21-24	1	159.19	69.50
13-16	2	236.64	75.70	17-20	4	334.08	63.86
21-24	6	302.69	73.09	17-20	4	334.08	63.86
21-24	6	302.69	73.09	13-16	8	193.22	23.10
17-20	12	6.50	6.39	13-16	8	193.22	23.10
17-20	12	6.50	6.39	21-24	24	2.69	0.74
	Amount to=	1386.379		21-24	24	2.69	0.74

Table 6. is based on the pharmacokinetic parameter of data in table 4 and the table 5

	Soluble	Crystalline
	Soluble	Crystalline	Dosage (μ g)	500	500
Dosage (mg/kg)	2.5	2.5	Dosage (μ g)	500	500
Dosage (mg/kg)	2.5	2.5	Half-life (hour)	0.5	9.4
C _max(ng/ml)	1172	334	Half-life (hour)	0.5	9.4
C _max(ng/ml)	1172	334	T _max(hour)	0.5	4
AUC(o-t)(ng/hr/ml)	2819	2472	T _max(hour)	0.5	4
AUC(o-t)(ng/hr/ml)	2819	2472	AUC(2)	2819	2508
T _90％(hour)	4	10	AUC(2)	2819	2508

Embodiment 17

Protamine sulfate is to the influence of the crystalline dissolving characteristic of hGH.Figure 17 has illustrated (the 85mM calcium acetate according to

embodiment

10,2% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6)) preparation the hGH crystal, after in the hGH calcium crystalloid solution that is pre-existing in, adding the protamine sulfate of specified rate, the meltage of cultivation after 1 hour in 37 ℃ of dissolving buffer.The ratio of HGH and protamine (mg: mg) be presented among Figure 17.Described figure explanation, the crystalline dissolving of protamine appreciable impact hGH.

Embodiment 18

With sodium acetate crystallization hGH.Here, from two kinds of storing solutions obtain the refrigerated a large amount of feedstock solution of hGH (rhGH) of soluble recombinant production-a kind of from escherichia coli (E.coli, Novartis) and another kind of from yeast (Lucky Gold).No matter its source produces the rhGH with the syncrystallization mutually and solubility characteristics from the rhGH separate analysis of escherichia coli and yeast stock solution.The rhGH feedstock solution that the about 3.3ml of 10DG-desalting column purification that use is supplied by BioRad (the 10-20mg/ml rhGH of supply in unknown buffer) thaws.Before sample loads, by (10mM, pH8.0) column scrubber is handled post with 30ml Tris-HCl.Load the rhGH sample then and allow it to enter described post by gravity.After discarding first 3ml eluent, add the 10mM Tris-HCl pH8.0 of another 5ml.Eluting is also collected the rhGH of 4.5ml desalination.Use Millipore concentrator (MWCO 10,000) under 3500rpm, to carry out then centrifugal concentrated 20-30 minute.The concentration of hGH is in the scope of 30mg/ml, as what pass through to measure in the trap (1mg/ml hGH A280=0.813 trap unit) of 280nm/0.813.By add deionized water, Tris-HCl (pH8.6), PEG-6000 and sodium acetate to they respectively in total solution final concentration be 100mM, 6% (v/v) and 500mM, the protein final concentration is 15mg/ml, to produce crystal.Mixed solution and down cultivated solution 12-16 hour leniently then at 33 ℃.Needle-like or rhabdolith and TEM imaging (Figure 18 A and 18B) have been obtained.Crystal length changes between about 2 to 25 μ m.Centrifugal and make crystal settling become piller after, extract supernatant and, crystallization yield is measured greater than 85%.Under the temperature between 33 ℃ and 15 ℃, also can form crystal, but the crystallization time that need increase also may cause the minimizing of yield.

Embodiment 19

The complexation of hGH sodium and Ionomer additive.Behind the mensuration crystallization yield (seeing embodiment 18), the rhGH sodium crystal is resuspended in mother solution (250mM NaOAc, 25mM Tris-HCl (pH8.6), 6%PEG-6000, and 7mg/ml protamine sulfate or 4.2mg/ml poly arginine) in, so that reach the final concentration of 21mg/ml rhGH sodium crystal.For rhGH and protamine sulfate, the ratio of protein and additive is that (mg: mg), for rhGH and poly arginine, the ratio of protein and additive was 5: 1 (mg: mg) in about 3: 1.These ratios are calculated as molar ratio, and this molar ratio is about 1: 1.715 for rhGH and protamine, is about 1: 0.587 for rhGH and poly arginine.Be resuspended in above-mentioned rhGH precipitation in the suitable mother solution equably and before the piller of centrifugal acquisition condensation, cultivate down and spend the night at 2-8 ℃.Remove supernatant and piller is resuspended in identical mother solution (not having the Ionomer additive) and storage under 4 ℃.

Additive concentration (mg/ml) by changing mother solution but still resuspension can obtain other rhGH: the polymeric additive ratio to the rhGH of 21mg/ml.For example, the concentration (10.5mg/ml) that can use the protamine sulfate that increases in the mother solution is to obtain 2: 1 rhGH in resuspended back: the ratio of additive.

Embodiment 20

With zinc acetate crystallization rhGH.With zinc acetate and acetone crystallization rhGH.The rhGH feedstock solution that the about 3.3ml of 10DG-desalting column purification (10-20mg/ml) that use is supplied by BioRad thaws.Before sample loads, by using 30ml Na ₂HPO ₄/ NaH ₂PO ₄(10mM, pH6.1) column scrubber is handled post.Load the rhGH sample then and allow it to enter described post by gravity.After discarding first 3ml eluent, add the 10mM Na of another 5.0ml ₂HPO ₄/ NaH ₂PO ₄PH6.1.Eluting is also collected the branch samples such as 4.5ml of desalination rhGH.Use Millipore concentrator (MWCO 10,000) under 3500rpm, to carry out then centrifugal concentrated 5-10 minute.The concentration of hGH is in the scope of 15mg/ml, as what pass through to measure in the trap (1mg/ml hGH A280=0.813 trap unit) of 280nm/0.813.Contain deionized water, 8.91mM Na by adding 400 μ l ₂HPO ₄/ NaH ₂PO ₄The mother solution of pH6.1,0.88mg/ml zinc acetate, 9.89% acetone, to 100 μ l at 10mM Na ₂HPO ₄/ NaH ₂PO ₄The 15mg/ml protein of the preparation (pH6.1) is to produce crystal.Mixed solution and down cultivated solution 24-48 hour leniently then at 15 ℃.Obtained the crystal of hexagon sample, width changes between about 2 to 25 μ m.Centrifugal and make crystal become pellets after, extract supernatant and, crystallization yield measured roughly 55%.

Embodiment 21

Complexation with calcium acetate crystallization hGH and hGH calcium and Ionomer additive (poly arginine).Here, obtain refrigerated a large amount of feedstock solution of the hGH (rhGH) of soluble recombinant production-a kind of from escherichia coli (Novartis), another kind of from two kinds of storing solutions from yeast (Lucky Gold).Use is by the about 3.5ml of 10DG-desalting column purification (the rhGH feedstock solution that Tris-HCl (10mM, pH8.0) 12mg/mlrhGH in) thaws of BioRad supply.Before sample loads, by (10mM, pH8.0) column scrubber is handled post with 30ml Tris-HCl.Then the rhGH sample is loaded and allow it to enter described post by gravity.After discarding first 3ml eluent, add the 10mM Tris-HCl pH8.0 of another 5.0ml.Eluting is also collected the rhGH of 4.5ml desalination.Use Millipore concentrator (MWCO10,000) to carry out centrifugal concentrated 20-30 minute then at 3500rpm.As by in the measurement of the trap (1mg/ml hGH A280=0.813 trap unit) at 280nm/0.813 place, the concentration of hGH is in the scope of 30mg/ml.By in rhGH 30mg/ml stock solution, adding 1MTris-HCl (pH8.6), 50%PEG-6000 and 1M calcium acetate, so that obtain the final concentration of 15mg/ml rhGH, 100mM Tris-HCl (pH8.6), 2% (v/v) PEG-6000 and 85mM calcium acetate, to produce crystal.Leniently mix then and be incorporated in 33 ℃ of following cultivation solution 12-16 hour.Obtain length range at about 2 acicular crystals of knowing 25 μ m.Extract supernatant and centrifugal and make crystal become piller after, crystallization yield is measured as greater than 85%.Under the temperature between 33 ℃ and 15 ℃, also can form described crystal, but need the crystallization time that increases and reduce yield.Behind the mensuration crystallization yield (seeing embodiment 18), rhGH calcium is resuspended in the preparation medium (5mMCaOAc, 100mM Tris-HCl (pH8.6), 6%PEG-6000 and 4.2mg/ml poly arginine), so that reach the final concentration of 21mg/ml rhGH calcium.For rhGH and poly arginine, the ratio of protein and additive is 5: 1 (mg: mg).For rhGH and poly arginine, calculating these ratios is about 10: 0.587 molar ratio.Be resuspended in above-mentioned rhGH piller in the suitable mother solution equably and before the piller of centrifugal acquisition condensation, cultivate down at 2-8 ℃ and spend the night.Remove supernatant and be resuspended in piller in the identical mother solution that does not have the ion-type additive and storage under 4 ℃.

Embodiment 22

The hGH and crystalline pharmacokinetics of hGH bivalent cation and the pharmacodynamic study that use the Sprague-Dawley subcutaneous rat to give.The purpose of this research is the sustained release of assessment hGH from hGH crystal suspension and the body weight that increases behind the subcutaneous implantation hGH crystal suspension in the Sprague-Dawley rat that hypophysectomizes.Research design is as follows:

Table 7. research design

Group # or test compound	Sample ^a	The sample explanation	The # of rat	Dosage level (milligram/rat/week)	Dosage regimen
Group # or test compound	Sample ^a	The sample explanation	The # of rat	Dosage level (milligram/rat/week)	Dosage regimen	1	The solubility medium	Buffer in the water for injection (WFI) ^b	3	--	100 μ l once a day, totally 7 days
2	Every day is soluble	Commercially available hGH among the WFI ^c(1.5mg/ml)	9	1.05	100 μ l once a day, totally 7 days	1	The solubility medium	Buffer in the water for injection (WFI) ^b	3	--	100 μ l once a day, totally 7 days
2	Every day is soluble	Commercially available hGH among the WFI ^c(1.5mg/ml)	9	1.05	100 μ l once a day, totally 7 days	3	Calcium acetate, PEG, protamine	See embodiment 10-(21 mg/ml, before injection the dilution and with mother solution 1 ^dThe gentle mixing 1: 1)	9	1.05	First day 100 μ l once
4	Calcium acetate, PEG	See embodiment 13-(21 mg/ml, before injection the dilution and with mother solution 1 ^dGentle mixing 1: 1, hGH: the ratio of protamine=5: 1)	9	1.05	First day 100 μ l once	3	Calcium acetate, PEG, protamine		9	1.05	First day 100 μ l once
4	Calcium acetate, PEG		9	1.05	First day 100 μ l once	5	Zinc acetate	See embodiment 20-(21 mg/ml, before injection the dilution and with mother solution 2 ^eThe gentle mixing 1: 1)	3	1.05	First day 100 μ l once
7	Medium	From group 3,4﹠5 ^fMother solution	3	--	First day 100 μ l once	5	Zinc acetate		3	1.05	First day 100 μ l once
7	Medium	From group 3,4﹠5 ^fMother solution	3	--	First day 100 μ l once	8	Medium	Mother solution from group 9	3	--	First day 100 μ l once
9	Calcium acetate, PEG, poly arginine	See embodiment 21-(21 mg/ml, before injection the dilution and with mother solution 1 ^dGentle mixing 1: 1, hGH: the ratio of poly arginine=12: 1)	9	1.05	First day 100 μ l once	8	Medium	Mother solution from group 9	3	--	First day 100 μ l once

^aIf all samples is stored in 4 ℃ and hope, be warmed to room temperature before the preparation medium that in d, defines and reach 30 minutes, and in the rat of quantity shown in being expelled to.

^bBuffer comprises 7.5mg/ml D-mannitol, 12mg/ml sucrose.

^cCommercially available hGH is from BesaGen Ltd., and Australia obtains.

^d5mM CaOAc, 4%PEG-6000,0.025M glycine (pH8.6), 15mg/ml D-mannitol, 60mg/ml sucrose.

^eZinc acetate, NaH ₂PO ₄(pH6.1), ethanol.

^fThe single preparation medium that defines among the use d is as the medium contrast of all these groups.

After the arrival, with 80 female Sprague-Dawley rats, body weight about 150 grams ± 25 grams and about 4-6 age in week, it is foster respectively that (roughly temperature is 21 ± 3 ℃ under controlled conditions, relative humidity 50 ± 20%, 12 little time and 12 hours dark, per hour 10-15 air exchange in each 24 hours period) and in whole research, allow it freely obtain pure water and laboratory grain.Before test, allow rat to conform a week.

In 80 rats, 48 quilts give hGH suspension according to table 7.At first day once or the continuous once a day seven days forms with single bolus infusion at the subcutaneous test compound that gives of back.Before injection, light and labelling were shaved up to three days in the injection site, after this optionally to promote injection.30-gauge * 8mm pin that use is connected 300 μ l syringes gives test compound.Before the suction syringe and again before administration, the roll-over test chemical compound does not cause foaming to guarantee suspension or solution homogeneity carefully.Volume injected approximately is every rat 0.1ml.

Gathered blood sample from organizing 1,5,7 and 8 in (every group has 3 rats) rat in the 4th, 32,96 and 168 hour after injection in first day.To further be divided into 3 groups again, every group of 3 rats from the blood sample of

group

2,3,4 and 9 (every group has 9 rats) rat.Here, from the rat of first subgroup, gathered blood sample in the 0.5th, 24,72 and 168 hour after injection in first day, from the rat of second subgroup, gathered blood sample at the 4th, 32,96 and 168 hour, and from the rat of the 3rd subgroup, gathered blood sample at the 8th, 48,120 and 168 hour.Usually do not anaesthetizing or CO by orbital sinus ₂/ O ₂It is hemorrhage that the rat of anesthesia goes up collection, and be collected in the BD Microtainer pipe with serum separating medium.Then in centrifugal sample under about 4 ℃ and recovery serum and chilled storage (-80 ℃ approximately) before mensuration hGH and IGF-1 level.

Compile blood serum sample then and under the situation of

group

2,3,4 and 9, form the timeline (timeline) of linear progression.By the variation of the meansigma methods of serum levels and described subgroup in the subgroup of some preset time, bioassay standard deviation.See Table 8-14 and Figure 19 A.When with special crystal formulations administration, show the serum levels (ng/ml) of rhGH.Getting blood with the corresponding concrete time point of administration to animal according to research approach.The result clearly illustrates, there are differences between the absorption of the crystal formulations (for example CaOAC and ZnOAC) of the crystalline solid (for example protamine and poly arginine) of complexation and non-complexation.

Table 8.hGH animal pharmaceuticals dynamics research result, group 2: every day is soluble

Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation
Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation	All	0	0	0
1-3	1	1262	18	All	0	0	0
1-3	1	1262	18	4-6	4	234	45
7-9	8	2	2	4-6	4	234	45
7-9	8	2	2	1-3	24	1218	258
4-6	32	3	1	1-3	24	1218	258
4-6	32	3	1	7-9	48	0	0
1-3	72	1098	40	7-9	48	0	0
1-3	72	1098	40	4-6	96	0	0
7-9	120	1355	337	4-6	96	0	0
7-9	120	1355	337	All	168	0	0
	Amount to=	5174		All	168	0	0

Table 9.hGH animal pharmaceuticals dynamics research result, group 3: calcium acetate, PEG

Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation
Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation	All	0	0.00	0.00
1-3	0.5	1927	771	All	0	0.00	0.00
1-3	0.5	1927	771	4-6	4	5204	1040
7-9	8	1409	881	4-6	4	5204	1040
7-9	8	1409	881	1-3	24	1	0.49
4-6	32	0.00	0.00	1-3	24	1	0.49
4-6	32	0.00	0.00	7-9	48	0.00	0.00
1-3	72	0.00	0.00	7-9	48	0.00	0.00
1-3	72	0.00	0.00	4-6	96	0.00	0.00
7-9	120	0.00	0.00	4-6	96	0.00	0.00
7-9	120	0.00	0.00	All	168	0.00	0.00
	Amount to=	8542		All	168	0.00	0.00

Table 10.hGH animal pharmaceuticals dynamics research result, group 4: calcium acetate, PEG, milt

Albumen

Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation
Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation	All	0	0.0000	0.00
1-3	0.5	0.00	0.00	All	0	0.0000	0.00
1-3	0.5	0.00	0.00	4-6	4	38	27
7-9	8	961	385	4-6	4	38	27
7-9	8	961	385	1-3	24	1468	357
4-6	32	192	73	1-3	24	1468	357
4-6	32	192	73	7-9	48	190	266
1-3	72	0	0	7-9	48	190	266
1-3	72	0	0	4-6	96	0	0
7-9	120	0	0	4-6	96	0	0
7-9	120	0	0	All	168	0	0
	Amount to=	2849		All	168	0	0

Table 11.hGH animal pharmaceuticals dynamics research result, group 5: zinc acetate

Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation
Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation	All	0	0.00	0.00
1	4	2417	767	All	0	0.00	0.00
1	4	2417	767	2	32	1	1
3	96	0	0	2	32	1	1
3	96	0	0	All	168	0	0
	Amount to=	2418		All	168	0	0

Table 12.hGH animal pharmaceuticals dynamics research result, group 9: calcium acetate, poly arginine

Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation
Animal	Get the blood time (hour)	Average hGH (ng/ml) in the serum	Standard deviation	All	0	0.00	0.00
1-3	1	502	75	All	0	0.00	0.00
1-3	1	502	75	4-6	4	846	102
7-9	8	1036	448	4-6	4	846	102
7-9	8	1036	448	1-3	24	634	462
4-6	32	543	168	1-3	24	634	462
4-6	32	543	168	7-9	48	407	169
1-3	72	9	0	7-9	48	407	169
1-3	72	9	0	4-6	96	0.00	0.00
7-9	120	0.00	0.00	4-6	96	0.00	0.00
7-9	120	0.00	0.00	All	168	0.00	0.00
	Amount to=	3980		All	168	0.00	0.00

Table 13. is based on the pharmacokinetic parameter of the data among the table 8-12

Group	Per 7 days dosage	C _max(ng/ml)	T _max(hour)
Group	Per 7 days dosage	C _max(ng/ml)	T _max(hour)	2: every day is soluble	6.7mg/kg	1262.70	1
3: calcium acetate, PEG	6.7mg/kg	5203.80	4	2: every day is soluble	6.7mg/kg	1262.70	1
3: calcium acetate, PEG	6.7mg/kg	5203.80	4	4: calcium acetate, PEG, protamine	6.7mg/kg	1468.47	8
5: zinc acetate	6.7mg/kg	2416.97	4	4: calcium acetate, PEG, protamine	6.7mg/kg	1468.47	8
5: zinc acetate	6.7mg/kg	2416.97	4	9: calcium acetate, poly arginine	6.7mg/kg	1036.63	8

Before injection in first day of research and got blood before the time in each successive morning of studying again, measure and write down the body weight of every rat.Therefore, by from each the continuous body weight of day (before the injection) deduct the 1st day body weight of (before the injection), calculate every group in weight increase or the minimizing of every rat.Calculate the weight average value of whole rats in group every day.These results are provided in the table 14.

The weight increase or alleviate (gram) every day of table 14.Sprague-Dawley rat

Group	The 1st day	The 2nd day	The 3rd day	The 4th day	The 5th day	The 6th day	The 7th day	The 8th day
Group	The 1st day	The 2nd day	The 3rd day	The 4th day	The 5th day	The 6th day	The 7th day	The 8th day	1	0	-3.31	1.00	-0.02	-4.78	-6.82	-9.02	-7.45
2	0	1.68	6.82	3.55	5.07	7.06	7.86	12.69	1	0	-3.31	1.00	-0.02	-4.78	-6.82	-9.02	-7.45
2	0	1.68	6.82	3.55	5.07	7.06	7.86	12.69	3	0	3.09	3.97	2.08	1.55	2.07	0.33	2.02
4	0	6.10	9.25	5.80	1.15	3.95	5.01	6.20	3	0	3.09	3.97	2.08	1.55	2.07	0.33	2.02
4	0	6.10	9.25	5.80	1.15	3.95	5.01	6.20	5	0	-0.09	1.40	-0.48	0.01	-1.41	-2.22	-0.95
7	0	-3.96	0.62	1.39	1.70	2.09	1.24	2.13	5	0	-0.09	1.40	-0.48	0.01	-1.41	-2.22	-0.95
7	0	-3.96	0.62	1.39	1.70	2.09	1.24	2.13	8	0	-2.18	-0.27	-1.42	0.85	-0.82	-1.16	0.61
9	0	4.17	8.45	8.81	8.09	9.10	7.31	10.00	8	0	-2.18	-0.27	-1.42	0.85	-0.82	-1.16	0.61

Table 14 has illustrated with the influences in seven days that give the commercially available hGH of daily dose with Figure 19 B and has compared, given single dose (the 1st day) influence in crystalline seven days of the present invention.For example, Figure 19 B confirms, has realized weight increase with the hGH crystal calcium of poly arginine complexation, this weight increase be comparable to a dose in the identical time period every day solvable dosage weight increase.In serum, in separately the release characteristic, be apparent that the poly arginine preparation of longer release and the more lasting velocity correlation of weight increase at the body weight that relatively increases and rhGH.

Embodiment 23

Comparison pharmacodynamic study in female young macaque (jiuvenile cynomologous monkeys).The purpose of this research is assessment when subcutaneous when giving female young macaque, crystallization recombinant human somatropin's (rhGH) interior medicine dynamics feature.Generate these data to set up the sustained release of crystallization rhGH in serum and the model of the weight increase of the function that discharges as crystallization rhGH.

The research design of table 15. primates research I

Group #	Sample	The giving of dosage (hour)	Dosage level (mg/kg)	Dose concentration (mg/ml)	Dosage volume (ml/kg)	Number of animals (female)
Group #	Sample	The giving of dosage (hour)	Dosage level (mg/kg)	Dose concentration (mg/ml)	Dosage volume (ml/kg)	Number of animals (female)	1	Every day is soluble ^a	0，24，48，72，9 6，120，144	0.8	3.2	0.25	4
2	Sodium acetate, PEG, poly arginine ^b	0	5.6	22.4	0.25	4	1	Every day is soluble ^a	0，24，48，72，9 6，120，144	0.8	3.2	0.25	4
2	Sodium acetate, PEG, poly arginine ^b	0	5.6	22.4	0.25	4	3	Sodium acetate, PEG, protamine ^b	0	5.6	22.4	0.25	4

^aCommercially available hGH (soluble, uncrystallized form) obtains and diafiltration among WFI from Novarits.Group 1 (positive control) accepted soluble hGH in each administration sky.

^bSee embodiment 18 and 19 about preparation.

^cAfter getting blood every day, send all dosage.

12 female young macaques are divided into three groups, every group has four animals, and give soluble rhGH (group 1), have the rhGH sodium crystal (group 2 is according to embodiment 18 and 19) of PEG and poly arginine or have the rhGH sodium crystal (group 3 is according to embodiment 18 and 19) of PEG and protamine.Will be when handling beginning body weight 2-6kg and age 4-7 the year monkey individually support in the Rotating Stainless Steel Cage that is being equipped with automatic water supply system or water bottle.(about 21 ± 3 ℃ of control animal room environments, 30-70% humidity, 12 little time and 12 hours dark in per 24 hours periods, and 12-20 air exchange per hour) and twice guaranteed coml primates of usefulness standard grain every day (Harlan Teklad Certified Primate Diet#2055C) feed monkey.

For measure and relatively give soluble rhGH (group 1), have the rhGH sodium crystal (group 2) of PEG and poly arginine or have the rhGH sodium crystal (group 3) of PEG and protamine after hGH and the serum-concentration of IGF-1, carry out the research of this primates.On the time shown in transfer and the above-mentioned table 15, before the administration, write down the body weight of all animals.In the-216 ,-120,0,2,4,6,8,10,24,48,72,96,120,144,168,192,216,240,264,288 and 312 days the morning, collect blood sample (approximately 1ml) from every animal through femoral vein, brachial veins or saphena.Blood collecting in serum separator tube, is at room temperature placed and was condensed allowing in 30-45 minute, and 3000rpm2-8 ℃ centrifugal 10 minutes down.Each blood serum sample is divided into 100 μ l sample aliquot and remaining sample aliquot, and they all are stored under-70 ± 10 ℃ before analysis.Typically, the 100 μ l sample aliquot that are used to rhGH mensuration are more little, and the remaining part that is used to IGF-1 mensuration is just big more.There are some exceptions in the volume that duplicates owing to needing.

Then the blood serum sample of collecting is carried out hGH concentration analysis (seeing Table 16).Make suitable dilution to dropping on the outer rhGH concentration of standard value range.Use the average background level of all values with every animal individual of acquisition primates GH.From the serum levels of the described subjects of each time point determining, deducting this every animal meansigma methods.Then with the correction value of each time point on average to obtain the modified mean of rhGH in the serum.Standard deviation basis of calculation error by using modified mean and divided by the square root of N=4 then.

Table 16. group 1 (every day is soluble), 2 (rhGH sodium/poly arginines)

RhGH level with 3 (rhGH sodium/protamines)

Time hour	Group soluble rhGH average every day of 1-(ng/ml)	Standard error	The group average rhGH sodium/poly arginine of 2-(ng/ml)	Standard error	The group average rhGH sodium/protamine of 3-(ng/ml)	Standard error
Time hour	Group soluble rhGH average every day of 1-(ng/ml)	Standard error	The group average rhGH sodium/poly arginine of 2-(ng/ml)	Standard error	The group average rhGH sodium/protamine of 3-(ng/ml)	Standard error	-216	4	7	-4	7	-2	1
-120	8	9	7	5	7	9	-216	4	7	-4	7	-2	1
-120	8	9	7	5	7	9	0	343	65	-4	3	-5	9
2	372	48	-6	6	1	13	0	343	65	-4	3	-5	9
2	372	48	-6	6	1	13	4	262	37	11	9	20	12
6	205	45	85	45	94	35	4	262	37	11	9	20	12
6	205	45	85	45	94	35	8	132	29	186	93	159	57
10	18	37	409	202	381	189	8	132	29	186	93	159	57
10	18	37	409	202	381	189	24	-14	7	404	17	333	37
48	-7	8	178	33	216	43	24	-14	7	404	17	333	37
48	-7	8	178	33	216	43	72	-3	10	77	35	86	18
96	-9	9	12	14	21	13	72	-3	10	77	35	86	18
96	-9	9	12	14	21	13	120	-11	6	6	13	2	11
144	-3	13	-6	10	3	13	120	-11	6	6	13	2	11
144	-3	13	-6	10	3	13	168	10	11	-2	4	-1	11
192	-11	10	3	2	0	7	168	10	11	-2	4	-1	11
192	-11	10	3	2	0	7	216	-13	9	18	12	9	6
240	1	5	18	14	31	12	216	-13	9	18	12	9	6
240	1	5	18	14	31	12	264	8	3	20	5	17	11
288	-15	8	1	4	17	11	264	8	3	20	5	17	11
288	-15	8	1	4	17	11	312	4	7	1	5	8	17

Annotate: the rhGH value is the meansigma methods from 4 animals, and it is by baseline adjustment, that is, value deducts baseline.Baseline is at t=-216 ,-120 and the meansigma methods of 0 one hour value.

Figure 20 A illustrated in the

group

1,2 and 3, behind baseline adjustment, as hour to be the level of rhGH in the serum of function of time of unit.

Table 17. is based on the summary of the pharmacokinetic parameter of data in the table 16

	Group 1 ^a	Group 2	Group 3
	Group 1 ^a	Group 2	Group 3	Dosage (mg)	3.2	22.4	22.4
Dosage (mg/kg)	0.8	5.6	5.6	Dosage (mg)	3.2	22.4	22.4
Dosage (mg/kg)	0.8	5.6	5.6	C _max(ng/ml)	372	409	381
T _max(hour)	2	10	10	C _max(ng/ml)	372	409	381
T _max(hour)	2	10	10	AUC(0-t) (ng.hr.kg/ml.mg)	4570	3503	3455
T _90％(hour)	20	74	77	AUC(0-t) (ng.hr.kg/ml.mg)	4570	3503	3455

^aWith commercially available hGH (soluble, uncrystallized form) diafiltration in WFI.Group 1 (positive control) accepted soluble hGH in the every day in 7 administration skies.

Above-mentioned data acknowledgement, for the hGH sodium crystal of poly arginine complexation, maximum hGH appears at the time (T in the serum _Max) be 10 hours, for the hGH sodium crystal of protamine complexation, be 10 hours, and, be 2 hours for soluble hGH.Suppose to use and send soluble hGH, the C that lists in the above-mentioned table 17 with the crystal of 1/7th dosage _MaxValue shows, when sending hGH with any of the crystal form of two kinds of complexations, significantly lowers initial serum-concentration spike.In addition, calculated solvable and the T crystal group _90%Value.The T of group 1 (soluble form) _90%Be 20 hours, and organize 2 and the group 3 (crystal forms of complexation) T _90%Be respectively 74 and 77 hours.These results clearly illustrate that the hGH level that the crystal form of complexation causes raising was kept than the obvious longer time of soluble form.

Except the serum-concentration of measuring hGH, also measured level as the IGF-1 of the function of time.By measuring the generation of IGF-1, determined the effect of rhGH.Following table 18 has been reported the IGF-1 concentration of animal among the group 1-3.Figure 20 B explanation, behind the baseline that deducts endogenous IGF-1 level, the ability that stimulates IGF-1 to discharge that the crystal formulations of complexation is verified, this ability is comparable to soluble using every day.These results in non-human primates show, can advantageously use preparation of the present invention to reach similar effect in the people.

Table 18. group (every day soluble, rhGH sodium/poly arginine

With rhGH sodium/protamine) the IGF-1 level

Time hour	Group soluble IGF-1 average every day of 1-(ng/ml)	Standard error	The average rhGH sodium of group 2-/poly arginine IGF-1 (ng/ml)	Standard error	The average rhGH sodium of group 3-/protamine IGF-1 (ng/ml)	Standard error
Time hour	Group soluble IGF-1 average every day of 1-(ng/ml)	Standard error		Standard error	The average rhGH sodium of group 3-/protamine IGF-1 (ng/ml)	Standard error	-216	-48	54	263	148	31	94
-120	-0.63	22	4	34	218	115	-216	-48	54	263	148	31	94
-120	-0.63	22	4	34	218	115	0	48	73	-268	158	-249	86
2	39	32	-160	146	-56	104	0	48	73	-268	158	-249	86
2	39	32	-160	146	-56	104	4	22	52	-344	191	-120	114
6	37	45	-244	189	-19	96	4	22	52	-344	191	-120	114
6	37	45	-244	189	-19	96	8	-22	9	-464	170	-66	119
10	106	63	-491	219	4	86	8	-22	9	-464	170	-66	119
10	106	63	-491	219	4	86	24	130	130	-106	278	223	214
48	446	59	164	244	191	164	24	130	130	-106	278	223	214
48	446	59	164	244	191	164	72	414	95	248	224	340	207
96	485	114	402	67	416	243	72	414	95	248	224	340	207
96	485	114	402	67	416	243	120	524	73	484	126	392	216
144	636	63	574	189	397	187	120	524	73	484	126	392	216
144	636	63	574	189	397	187	168	636	82	415	191	240	176
192	438	71	356	136	227	153	168	636	82	415	191	240	176
192	438	71	356	136	227	153	216	288	87	155	108	117	146
240	210	69	197	57	93	144	216	288	87	155	108	117	146
240	210	69	197	57	93	144	264	161	67	88	82	85	167
288	222	113	243	95	201	208	264	161	67	88	82	85	167
288	222	113	243	95	201	208	312	178	175	79	120	86	149

Annotate: the IGF-1 value is reported as the meansigma methods of being calculated by 4 animals, and it is by baseline adjustment, that is, value deducts baseline.Baseline is at t=-216 ,-120 and the meansigma methods of 0 one hour value.

Embodiment 24

With the comparison pharmacodynamic study of different protamine ratios in female young macaque.The purpose of this research is assessment when subcutaneous when giving female young macaque, crystallization recombinant human somatropin's (rhGH) interior medicine dynamics feature.Generate these data with the ratio of research hGH sodium and protamine to crystallization rhGH in serum sustained release and as the influence of the weight increase of the function of crystallization rhGH release.

The experimentation of table 19. primates research II

Group #	Sample	The giving of dosage (hour)	Dosage level (mg/kg)	Dose concentration (mg/ml)	Dose volume (ml/kg)	Number of animals (female)
Group #	Sample	The giving of dosage (hour)	Dosage level (mg/kg)	Dose concentration (mg/ml)	Dose volume (ml/kg)	Number of animals (female)	1	Every day is soluble ^a	0，24，48，72， 96，120，144	0.8	3.2	0.25	4
2	Sodium acetate, PEG, protamine (3: 1) ^b	0	5.6	22.4	0.25	4	1	Every day is soluble ^a	0，24，48，72， 96，120，144	0.8	3.2	0.25	4
2	Sodium acetate, PEG, protamine (3: 1) ^b	0	5.6	22.4	0.25	4	3	Sodium acetate, PEG, protamine (2: 1) ^b	0	5.6	22.4	0.25	4

^bSee embodiment 18 and 19 about preparation.

^cAfter getting blood every day, send all dosage.

In primates research II, to study the female young macaque of 12 described in the I primates and be divided into three groups, every group has four animals, and gives soluble rhGH (group 1), has rhGH sodium crystal (3: 1 rhGH: protamine) (group 2) (embodiment 18 and 19) or have rhGH sodium crystal (2: 1 rhGH: protamine) (group 3) (embodiment 18 and 19) of PEG and protamine of PEG and protamine.Will be when handling beginning body weight 2-6kg and age 4-7 the year monkey individually support in the Rotating Stainless Steel Cage that is being equipped with automatic water supply system or water bottle.(about 21 ± 3 ℃ of control animal room environments, 30-70% humidity, 12 little time and 12 hours dark in per 24 hours periods, and 12-20 air exchange per hour) and twice guaranteed coml primates of usefulness standard grain every day (Harlan TekladCertified Primate Diet #2055C) feed monkey.

For measure and relatively give soluble rhGH (group 1), have PEG and protamine the rhGH sodium crystal (3: 1 rhGH: protamine) (group 2) and have PEG and protamine the rhGH sodium crystal (2: 1 rhGH: protamine) hGH after (group 3) and the serum-concentration of IGF-1, carry out this primates and study.Before time administration shown in transfer and the above-mentioned table 19, write down the body weight of all animals.In the-144 ,-120 ,-96 ,-72 ,-48 ,-24,0,2,4,6,8,10,24,48,72,96,120,144,168,192,216,240,264,288 and 312 days the morning, collect blood sample (approximately 1ml) from every animal through femoral vein, brachial veins or saphena.Blood collecting in serum separator tube, is at room temperature placed and was condensed allowing in 30-45 minute, and 3000rpm 2-8 ℃ centrifugal 10 minutes down.Each blood serum sample is divided into 100 μ l sample aliquot and remaining sample aliquot, and they all are stored under-70 ± 10 ℃ before detection.

HGH concentration (ng/ml) (seeing Table the data in 20) in the blood serum sample that analysis and baseline correction are collected.Note, make suitable dilution dropping on the outer rhGH concentration of standard value range.Use the average background level of all values then with every animal individual of acquisition primates hGH.From the serum levels of the described subjects of each time point determining, deducting this every animal meansigma methods.Then with the correction value of each time point on average to obtain the modified mean of rhGH in the serum.Standard deviation basis of calculation error by using modified mean and divided by the square root of N=4 then.

Table 20. group 1 (every day is soluble), 2 (rhGH sodium/protamine (3: 1))

RhGH level with 3 (rhGH sodium/protamine (2: 1))

Time (hour)	Group soluble rhGH average every day of 1-(ng/ml)	Standard error	Group 2 average rhGH sodium/protamines (3: 1), (ng/ml)	Standard error	The group average rhGH sodium/protamine of 3-(2: 1) (ng/ml)	Standard error
Time (hour)	Group soluble rhGH average every day of 1-(ng/ml)	Standard error	Group 2 average rhGH sodium/protamines (3: 1), (ng/ml)	Standard error	The group average rhGH sodium/protamine of 3-(2: 1) (ng/ml)	Standard error	-144	-14	6	-13	6	34	22
-120	-14	5	-9	5	10	6	-144	-14	6	-13	6	34	22
-120	-14	5	-9	5	10	6	-96	18	12	48	23	28	43
-72	-1	5	-5	6	3	9	-96	18	12	48	23	28	43
-72	-1	5	-5	6	3	9	-48	-9	5	-2	5	14	6
-24	-2	9	-14	7	-3	10	-48	-9	5	-2	5	14	6
-24	-2	9	-14	7	-3	10	0	21	9	-4	9	1	5
2	312	47	8	10	-29	16	0	21	9	-4	9	1	5
2	312	47	8	10	-29	16	4	401	57	77	32	13	8
6	186	16	172	62	89	52	4	401	57	77	32	13	8
6	186	16	172	62	89	52	8	157	29	330	104	222	108
10	172	24	456	109	364	142	8	157	29	330	104	222	108
10	172	24	456	109	364	142	24	3	4	316	29	372	74
48	8	7	153	47	128	15	24	3	4	316	29	372	74
48	8	7	153	47	128	15	72	6	6	116	81	30	23
96	-2	5	42	15	13	21	72	6	6	116	81	30	23
96	-2	5	42	15	13	21	120	14	3	22	22	22	14
144	16	16	8	12	-13	11	120	14	3	22	22	22	14
144	16	16	8	12	-13	11	168	14	8	7	6	-21	13
192	2	7	-4	7	3	22	168	14	8	7	6	-21	13
192	2	7	-4	7	3	22	216	11	9	6	14	-29	19
240	27	19	14	9	-4	8	216	11	9	6	14	-29	19
240	27	19	14	9	-4	8	264	-1	6	35	21	-19	18
288	-2	9	2	5	-32	18	264	-1	6	35	21	-19	18
288	-2	9	2	5	-32	18	312	-0.58	9	10	6	-21	18

Annotate: the rhGH value is reported as the meansigma methods of being calculated by 4 animals, and it is by baseline adjustment, that is, value deducts baseline.Baseline is at t=-144 ,-120 ,-96 ,-72 ,-48 ,-24 and the meansigma methods of 0 one hour value.

Figure 21 A illustrated in the

group

Table 21. is based on the summary of the pharmacokinetic parameter of data in the table 20

	Group 1 ^a	Group 2	Group 3
	Group 1 ^a	Group 2	Group 3	Dosage (mg)	3.2	22.4	22.4
Dosage (mg/kg)	0.8	5.6	5.6	Dosage (mg)	3.2	22.4	22.4
Dosage (mg/kg)	0.8	5.6	5.6	C _max(ng/ml)	401	456	380
T _max(hour)	4	10	24	C _max(ng/ml)	401	456	380
T _max(hour)	4	10	24	AUC(0-t) (ng.hr.kg/ml.mg)	4432	3669	2893
T ^90％-(hour)	20	119	72	AUC(0-t) (ng.hr.kg/ml.mg)	4432	3669	2893

These data acknowledgements, for the hGH crystal of protamine (3: 1) complexation, the time that maximum hGH appears in the serum is 10 hours, for the hGH crystal of protamine (2: 1) complexation, is 24 hours, and for soluble hGH, is 4 hours.Suppose to use and send soluble hGH, the C that lists in the above-mentioned table 22 with the crystal of 1/7th dosage _MaxValue shows, when sending hGH with any form in the crystallization of two kinds of complexations, significantly lowers maximum serum-concentration.The T of group 1 (soluble form) _90%Be 20 hours, and organize 2 and the group 3 (crystal forms of complexation) T _90%Be respectively 119 and 72 hours.These results clearly illustrate that the hGH level that the crystal form of complexation causes raising was kept than the obvious longer time of soluble form.

Except the serum-concentration of measuring hGH, also measured level as the IGF-1 of the function of time.By measuring the generation of IGF-1, determined the effect of rhGH.Following table 22 has been reported the IGF-1 concentration of animal among the group 1-3.Figure 21 B explanation, behind the baseline that deducts endogenous IGF-1 level, the crystal formulations of complexation can stimulate IGF-1 to discharge, and this ability is comparable to soluble using every day.These non-human primates result shows, can advantageously use preparation of the present invention to cause similar effect in the people.

Table 22. group 1 (every day is soluble), 2 (rhGH sodium/protamine (3: 1))

IGF-1 level with 3 (rhGH sodium/protamine (2: 1))

Time (hour)	1-is soluble average every day for group, and IGF-1 (ng/ml)	Standard error	The group average rhGH sodium/protamine of 2-(3: 1), IGF-1 (ng/ml)	Standard error	The group average rhGH sodium/protamine of 3-(2: 1), IGF-1 (ng/ml)	Standard error
Time (hour)	1-is soluble average every day for group, and IGF-1 (ng/ml)	Standard error		Standard error		Standard error	-144	49	92	123	60	137	134
-120	-61	19	88	42	57	98	-144	49	92	123	60	137	134
-120	-61	19	88	42	57	98	-96	-116	27	-178	11	-130	13
-72	-33	59	-69	40	-52	43	-96	-116	27	-178	11	-130	13
-72	-33	59	-69	40	-52	43	-48	24	47	-102	100	-10	64
-24	22	46	-56	85	-83	78	-48	24	47	-102	100	-10	64
-24	22	46	-56	85	-83	78	0	115	71	194	106	82	107
2	-15	71	-6	79	51	95	0	115	71	194	106	82	107
2	-15	71	-6	79	51	95	4	-6	96	45	42	-10	94
6	48	106	38	63	91	74	4	-6	96	45	42	-10	94
6	48	106	38	63	91	74	8	10	119	97	47	88	78
10	38	106	-37	56	57	75	8	10	119	97	47	88	78
10	38	106	-37	56	57	75	24	200	160	20	48	150	107
48	99	90	85	68	342	97	24	200	160	20	48	150	107
48	99	90	85	68	342	97	72	505	391	100	155	278	105
96	328	202	363	161	289	122	72	505	391	100	155	278	105
96	328	202	363	161	289	122	120	329	224	294	89	261	136
144	279	282	210	81	86	103	120	329	224	294	89	261	136
144	279	282	210	81	86	103	168	591	266	424	184	219	104
192	259	185	169	163	131	50	168	591	266	424	184	219	104
192	259	185	169	163	131	50	216	127	152	55	107	-8	67
240	54	153	-54	141	-64	72	216	127	152	55	107	-8	67
240	54	153	-54	141	-64	72	264	4	132	-165	103	-50	49
288	-16	105	-208	122	-43	75	264	4	132	-165	103	-50	49
288	-16	105	-208	122	-43	75	312	11	100	-77	207	30	77

Annotate: the IGF-1 value is reported as the meansigma methods of being calculated by 4 animals, and it is by baseline adjustment, that is, value deducts baseline.Baseline is at t=-144 ,-120 ,-96 ,-72 ,-48 ,-24 and the meansigma methods of 0 one hour value.

Embodiment 25

By single or every day subcutaneous injection give the human growth hormone's that the male rat hypophysectomize gives pharmacodynamic study.The purpose of this research is relatively when once or continuous seven days of every day during the subcutaneous male Wistar rat that hypophysectomizes, the effect of different hGH preparations.Research design is as follows:

Table 23. research design-sample explanation

Group # or test compound	Sample ^a	The sample explanation
Group # or test compound	Sample ^a	The sample explanation	1	Every day soluble medium-false hypophysectomy	(not having hGH) 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH ₂PO ₄(pH6.5)
2	Every day soluble medium-low dosage	(not having hGH) 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH ₂PO ₄(pH6.5)	1	Every day soluble medium-false hypophysectomy
2	Every day soluble medium-low dosage		3	Every day soluble medium-high dose	(not having hGH) 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH ₂PO ₄(pH6.5)
4	Every day is soluble-low dosage	0.71mg/ml rhGH, 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH ₂PO ₄(pH6.5)	3	Every day soluble medium-high dose
4	Every day is soluble-low dosage		5	Every day is soluble-high dose	1.0mg/ml rhGH, 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH ₂PO ₄(pH6.5)
6	Soluble single bolus infusion high dose	3.5mg/ml rhGH, 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH ₂PO ₄(pH6.5)	5	Every day is soluble-high dose
6	Soluble single bolus infusion high dose		7	Poly arginine crystal-high dose	18.7mg/ml rHGH crystal, 250mM NaOAc, 6%PEG-6000,25 mM Tris-HCl (pH8.6), 3.6mg/ml poly arginine HCl (rhGH: the mol ratio of poly arginine is 1: 0.587)
8	Medium contrast-protamine crystal	250mM NaOAc, 6%PEG-6000,25mM Tris-HCl (pH8.6), 0.75mg/ml protamine sulfate	7	Poly arginine crystal-high dose
8	Medium contrast-protamine crystal		9	Protamine crystal-low dosage	3.3mg/ml rHGH crystal, 250mM NaOAc, 6%PEG-6000,25mM Tris-HCl (pH8.6), 0.75mg/ml protamine sulfate (rhGH: the mol ratio of poly arginine is 1: 1.715)
10	Protamine crystal-high dose	18.7mg/ml rHGH crystal, 250mM NaOAc, 6%PEG-6000,25 mM Tris-HCl (pH8.6), 4mg/ml protamine sulfate (rhGH: the mol ratio of poly arginine is 1: 1.715)	9	Protamine crystal-low dosage
10	Protamine crystal-high dose		11	Medium contrast-poly arginine crystal	250mM NaOAc, 6%PEG-6000,25mM Tris-HCl (pH8.6), 3.6mg/ml poly arginine-HCl
12	Medium contrast-single bolus infusion	16.7mg/ml the D-mannitol, 26.7mg/ml sucrose, 50mM NaH ₂PO ₄(pH6.5)	11	Medium contrast-poly arginine crystal

^aUse WFI under aseptic condition, to prepare whole samples.After solution transferred to their final volume separately, with 0.22 μ m filter filtration supports and soluble hGH sample.

Table 24. research design-administration

Group # or test compound	Administration level (mg/kg)	Administration concentration (mg/ml)	Administration volume (μ l)	Dosage regimen	Size of animal (male)
Group # or test compound	Administration level (mg/kg)	Administration concentration (mg/ml)	Administration volume (μ l)	Dosage regimen	Size of animal (male)	1	0	0	200	Administration 7 every days	13
2	0	0	20	Administration 7 every days	11	1	0	0	200	Administration 7 every days	13
2	0	0	20	Administration 7 every days	11	3	0	0	80	Administration 7 every days	11
4	0.143	0.71	20	Administration 7 every days	11	3	0	0	80	Administration 7 every days	11
4	0.143	0.71	20	Administration 7 every days	11	5	0.8	1	80	Administration 7 every days	12
6	5.6	3.5	160	The 1st day	11	5	0.8	1	80	Administration 7 every days	12
6	5.6	3.5	160	The 1st day	11	7	5.6	18.7	30	The 1st day	12
8	0	0	30	The 1st day	11	7	5.6	18.7	30	The 1st day	12
8	0	0	30	The 1st day	11	9	1	3.3	30	The 1st day	12
10	5.6	18.7	30	The 1st day	12	9	1	3.3	30	The 1st day	12
10	5.6	18.7	30	The 1st day	12	11	0	0	30	The 1st day	11
12	0	0	30	The 1st day	11	11	0	0	30	The 1st day	11

After the arrival, with 138 female Wistar rats, the about 90-100 of body weight gram and approximately 25-30 days big, grouping is foster, and (roughly temperature is 23 ± 3 ℃ under controlled conditions, relative humidity 30-70%, 12 little time and 12 hours dark, per hour 10-15 air exchange in each 24 hours period) and in whole research, allow it freely obtain pure water and laboratory grain.Before test, allow rat to conform for two weeks.

According to the concentration in the table 24, volume and 138 rat samples of relieve pain.Once or continuous once a day seven days with the single bolus infusion form at the back the subcutaneous test compound that gives.Before administration, light and labelling were shaved up to three days in the injection site, after this optionally to promote injection.30-gauge * 8mm pin that use is connected on the 300 μ l syringes gives test compound.Before the suction syringe and again before administration, the roll-over test chemical compound does not cause foaming to guarantee suspension or solution homogeneity carefully.

During-3 weeks and-2 weeks weekly twice and from-7 days to 14 day every day measure and the record weight increase.During administration, rat body weight approximately is 100g ± 10%.In Figure 22 and 23, provide and in table 25 and 26, summed up the percentage result of induced growth." high dose " expression 5.6mg/kg/ week in the table 25.Data declaration single injection rhGH: poly arginine (group 7, embodiment 18 and 19) or rhGH: protamine (group 9 and 10, embodiment 18 and 19) crystal comparison of rat body weight increase during identical seven days with injection contrast every day (group 1 does not have hGH) or soluble hGH sample (group 4 and 5) during seven days.Group 1, false hypophysectomy rat has shown the normal growth during seven days.In addition, a shot in seven days gives rhGH: the rat of poly arginine (group 7) has higher induced growth percentage ratio than those rats that give soluble hGH (group 5) seven day every day.These presentation of results, the same effective according to hGH crystal of the present invention and a preparation and an every day in week giving soluble rhGH.

Table 25. is 8 days inductive weight increase in the rat that hypophysectomizes

Group # or test compound	The sample explanation	8 days inductive weight increase
Group # or test compound	The sample explanation	8 days inductive weight increase	1	False hypophysectomy	22％
7	Poly arginine crystal-high dose	21％	1	False hypophysectomy	22％
7	Poly arginine crystal-high dose	21％	5	Every day soluble high dose	20％
4	Every day soluble low dosage	11％	5	Every day soluble high dose	20％
4	Every day soluble low dosage	11％	10	Protamine crystal-high dose	5％
6	Every day soluble single bolus infusion high dose	2％	10	Protamine crystal-high dose	5％
6	Every day soluble single bolus infusion high dose	2％	9	Protamine crystal-low dosage	2％

Table 26. in the rat that hypophysectomizes every day inductive weight increase

Group	My god
	My god								0	1	2	3	4	5	6	7
	1	0	3	7	10	13	15	19	0	1	2	3	4	5	6	7	22
7	1	0	3	7	10	13	15	19	0	4	10	15	18	19	20	21	22
7	4	0	2	3	4	5	8	11	0	4	10	15	18	19	20	21	11
5	4	0	2	3	4	5	8	11	0	3	6	10	12	15	18	20	11
5	10	0	5	9	7	5	6	6	0	3	6	10	12	15	18	20	5
6	10	0	5	9	7	5	6	6	0	3	3	2	2	2	3	2	5
6	9	0	4	4	2	0	2	2	0	3	3	2	2	2	3	2	2

Embodiment 26

With sodium acetate and protamine sulfate crystallization hGH.Here, derive from escherichia coli (Novartis) from two kinds of storing solutions-a kind of, another kind derives from freezing a large amount of feedstock solution that yeast (Lucky Gold) obtains the hGH (rhGH) of soluble recombinant production.No matter its source, the separate analysis that derives from rhGH in escherichia coli and the yeast stock solution produces the rhGH with the syncrystallization mutually and solubility characteristics.The rhGH feedstock solution that the about 3.3ml of 10DG desalting column purification (10-20mg/ml) that use is supplied by BioRad thaws.Before sample loads, by (10mM, pH8.0) post is handled in washing with 30ml Tris-HCl.Load the rhGH sample then and allow it to enter described post by gravity.After discarding first 3ml eluent, add the 10mM Tris-HCl pH8.0 of another 5.0ml.Eluting is also collected the desalination rhGH of 4.5ml.Use Millipore concentrator (MWCO 10,000) under 3500rpm, to carry out then centrifugal concentrated 20-30 minute.As by in the measurement of the trap (1mg/mlhGH A280=0.813 trap unit) at 280nm/0.813 place, the concentration of hGH is in the scope of 30mg/ml.By add deionized water, Tris-HCl (pH8.6), PEG-4000, protamine sulfate and sodium acetate to they in total solution, be respectively the final concentration of 100mM, 6% (v/v), 2mg/ml and 500mM, final protein concentration is 15mg/ml, generates crystal.Mixed solution and down cultivated solution 12-16 hour leniently then at 33 ℃.Having obtained length is the acicular crystal of about 2 to 25 μ m.Centrifugal and make crystal become piller after, extract supernatant and, crystallization yield is measured as greater than 90%.

Embodiment 27

With sodium acetate and poly arginine HCl crystallization hGH.Here, derive from escherichia coli (Novartis) from two kinds of storing solutions-a kind of, another kind derives from yeast (Lucky Gold), obtains freezing a large amount of feedstock solution of the hGH (rhGH) of soluble recombinant production.No matter its source, the separate analysis that derives from rhGH in escherichia coli and the yeast stock solution produces the rhGH with the syncrystallization mutually and solubility characteristics.The rhGH feedstock solution that the about 3.3ml of 10DG desalting column purification (10-20mg/ml) that use is supplied by BioRad thaws.Before sample loads, by (10mM, pH8.0) post is handled in washing with 30ml Tris-HCl.Load the rhGH sample then and allow it to enter described post by gravity.After discarding first 3ml eluent, add the 10mM Tris-HCl pH8.0 of another 5.0ml.Eluting is also collected the desalination rhGH of 4.5ml.Use Millipore concentrator (MWCO 10,000) under 3500rpm, to carry out then centrifugal concentrated 20-30 minute.As by in the measurement of the trap (1mg/mlhGH A280=0.813 trap unit) at 280nm/0.813 place, the concentration of hGH is in the scope of 30mg/ml.By add deionized water, Tris-HCl (pH8.6), PEG-4000, poly arginine HCl and sodium acetate to they in total solution, be respectively the final concentration of 100mM, 2% (v/v), 2mg/ml and 500mM, final protein concentration is 15mg/ml, generates crystal.Mixed solution and down cultivated solution 12-16 hour leniently then at 33 ℃.Having obtained length is the acicular crystal of about 2 to 25 μ m.Centrifugal and make crystal become piller after, extract supernatant and, crystallization yield is measured as greater than 90%.

Although with illustrating and being to understand the clearly embodiment of purpose, with some details aforementioned invention has been described, but according to instruction of the present invention, those skilled in the art is apparent that easily, do not depart from the application's disclosure spirit or scope of (comprising appended embodiment), can make some variation and modification the present invention.

Claims

1. the calcium crystal of human growth hormone or human growth hormone's derivant.

2. the monovalent cation crystal of human growth hormone or human growth hormone's derivant.

3. the protamine crystal of human growth hormone or human growth hormone's derivant.

4. the poly arginine crystal of human growth hormone or human growth hormone's derivant.

5. according to the monovalent cation crystal of claim 2, wherein said monovalent cation is selected from the group of being made up of lithium, sodium, potassium and ammonium.

6. according to the monovalent cation crystal of claim 5, wherein said monovalent cation is a sodium.

7. according to claim 1,2,3 or 4 each crystal, wherein provide hGH serum-concentration in the body to the described crystal of mammiferous single-dose in described mammal, this serum-concentration is selected from the group of being made up of following:

(a) at about 0.3ng/ml to about 2, between the 500ng/ml hGH;

(b) at about 0.5ng/ml to about 1, between the 000ng/ml hGH;

(c) at about 1ng/ml between about 100ng/ml hGH, through the time period that being selected from by following every group of forming:

(i) using between back about 0.5 hour and about 40 days;

(ii) using between back about 0.5 hour and about 10 days;

(iii) using between back about 0.5 hour and about 7 days; And

(iv) using between back about 0.5 hour and about 1 day.

8. according to claim 1,2,3 or 4 each crystal, wherein in described mammal, provide IGF-1 serum rising in the body to the described crystal of mammiferous single-dose, more than the baseline IGF-1 level of this serum rising before described using, be selected from the group of forming by following:

(a) at about 5ng/ml to about 2, between the 500ng/ml; And

(b) at about 100ng/ml to about 1, between the 000ng/ml,

Through being selected from time period by following every group of forming:

(i) using between back about 0.5 hour and about 40 days;

(ii) using between back about 0.5 hour and about 7 days.

9. according to claim 1,2,3 or 4 each crystal, wherein compare with the relative bioavailability of the soluble hGH of same dose that sends by identical route of administration, described crystal has at least 50% or more relative bioavailability, wherein by described soluble hGH and described crystalline overall in the AUC of hGH serum-concentration measure described bioavailability.

10. according to the crystal of claim 7 or 8, wherein said mammal is the people.

11. according to the crystal of claim 1, wherein said crystal comprises about 1 monomer to about 500 everyone growth hormone of calcium molecule or human growth hormone's derivant.

12. according to the crystal of claim 2, wherein said crystal comprises about 1 monomer to about 500 everyone growth hormone of monovalent cation or human growth hormone's derivant.

13. according to the crystal of claim 1, wherein said crystal comprises the calcium salt that is selected from the group of being made up of calcium acetate, calcium chloride, calcium sulfate and calcium gluconate.

14. according to the crystal of claim 13, wherein said calcium salt is a calcium acetate.

15. according to the crystal of claim 6, wherein said crystal comprises the sodium salt that is selected from the group of being made up of sodium citrate, sodium phosphate and sodium acetate.

16. according to the crystal of claim 15, wherein said sodium salt is a sodium acetate.

17. a compositions, said composition comprise the crystal and the excipient of human growth hormone or human growth hormone's derivant, wherein said crystal is selected from the group of being made up of following:

(a) the calcium crystal of human growth hormone or human growth hormone's derivant;

(b) the monovalent cation crystal of human growth hormone or human growth hormone's derivant;

(c) the protamine crystal of human growth hormone or human growth hormone's derivant;

(d) the poly arginine crystal of human growth hormone or human growth hormone's derivant.

18. according to the compositions of claim 17, wherein said crystal and described excipient are with hGH: excipient is to be present in the described compositions to about 1: 0.125 mol ratio in about 1: 10.

19. according to the compositions of claim 17, wherein said excipient is selected from the group of being made up of aminoacid, salt, alcohol, carbohydrate, protein, lipid, surfactant, polymer, polyamino acid and their mixture.

20. according to the compositions of claim 19, wherein said excipient is selected from the group of being made up of protamine, polyvinyl alcohol, cyclodextrin, glucosan, calcium gluconate, polyamino acid, Polyethylene Glycol, dendrimers, poly ornithine, polymine, chitosan and their mixture.

21. according to the compositions of claim 20, wherein said excipient is selected from the group of being made up of protamine, poly arginine, Polyethylene Glycol and their mixture.

22. according to the compositions of claim 17, wherein human growth hormone or the concentration of human growth hormone's derivant in described compositions are selected from the group of being made up of following:

(a) about 0.1 and about 100mg/ml between;

(b) about 1 and about 100mg/ml between; And

(c) about 10 and about 100mg/ml between.

23. a treatment has the mammiferous method of obstacle, described obstacle lacks relevant with the human growth hormone or is improved by the personnel selection growth hormone therapy, this method comprise the claim 1,2 that gives described mammal treatment effective dose, 3 or 4 each crystal or the step of the compositions of claim 17.

24. a method of in mammal, inducing weight increase, this method comprise the claim 1,2 that gives described mammal treatment effective dose, 3 or 4 each crystal or the step of the compositions of claim 17.

25. according to the method for claim 24, wherein said mammal is HPX rat and uses behind the described crystal in described rat inductive weight increase between 5% and about 40% by injection weekly.

26. according to the method for claim 23, wherein said obstacle is selected from the group of being made up of adult's growth hormone deficiency, children growth hormonoprivia, Pu-Wei syndrome, Turner syndrome, short bowel syndrome, chronic renal insufficiency, spontaneous short stature, dwarfism, hypopituitarism dwarfism, osteanagenesis, female sterile disease, intrauterine growth retardation, AIDS related cachexia, segmental enteritis and burn.

27. according to the method for claim 26, wherein said obstacle be infant growth hormonoprivia and described method in described mammal, cause about 7 and about 11cm between the annual speed of growth.

28. according to the method for claim 23 or 24, wherein through port approach, parenteral approach, subcutaneous route or intramuscular approach give described crystal of described mammal or compositions.

29. according to the method for claim 28, wherein the subcutaneous route that has more than or equal to the pin of 27 gauge by use gives described crystal of described mammal or compositions.

30., wherein give described crystal of described mammal or compositions by Needleless injection or change dosage infusion pump according to the method for claim 23 or 24.

31., wherein give described crystal of described mammal or compositions by the time scheme that is selected from by the following group of forming according to the method for claim 23 or 24:

(a) made an appointment with once in per three days;

(b) week approximately once;

(c) per two weeks approximately once; And

(d) made an appointment with once in every month.

32. according to the method for claim 23 or 24, wherein said mammal is the people.

33. calcium crystal, monovalent cation crystal, protamine crystal or the crystalline method of poly arginine of producing human growth hormone or human growth hormone's derivant, this method comprises the steps:

(a) solution with human growth hormone or human growth hormone's derivant mixes with crystallization solution, and described crystallization solution comprises calcium salt or monovalent cation crystalline salt and Ionomer, and wherein said Ionomer is protamine or poly arginine; And

(b) under the temperature between about 4 ℃ and about 37 ℃, cultivate described crystalloid solution greater than about 12 hours, till the calcium crystal, monovalent cation crystal, protamine crystal or the poly arginine crystal that produce human growth hormone or human growth hormone's derivant.

34. according to the method for claim 33, wherein said Ionomer is a polylysine.

35. according to the method for claim 33, wherein said Ionomer is any two or more the mixture in protamine, poly arginine and the polylysine.

36. calcium crystal or the crystalline method of monovalent cation of producing human growth hormone or human growth hormone's derivant, this method comprises the steps:

(a) solution with human growth hormone or human growth hormone's derivant mixes with the crystallization buffer to produce crystallization solution;

(b) in described crystallization solution, add deionized water;

(c) in described crystallization solution, add precipitant;

(d) in described crystallization solution, add calcium salt or monovalent cation salt;

(e) under the temperature between about 10 ℃ and about 40 ℃, cultivate described crystallization solution about 2 to about 168 hours, till the calcium crystal or monovalent cation crystal that form human growth hormone or human growth hormone's derivant; And

(f) in the calcium crystal of described human growth hormone or human growth hormone's derivant or monovalent cation crystal, add Ionomer or ion-type micromolecule.

37. calcium crystal or the crystalline method of monovalent cation of producing human growth hormone or human growth hormone's derivant, this method comprises the steps:

(b) in described crystallization solution, add deionized water;

(c) in described crystallization solution, add ion-type micromolecule or Ionomer;

(e) under the temperature between about 10 ℃ and about 40 ℃, cultivate described crystallization solution about 2 to about 168 hours, till the calcium crystal or monovalent cation crystal that form human growth hormone or human growth hormone's derivant.

38. according to the method for claim 37, wherein, step (b) afterwards and step (c) before, described method comprises in described crystallization solution the step of adding precipitant.

39. according to each method in the claim 33,36 or 37, wherein said calcium salt is selected from the group of being made up of calcium acetate, calcium chloride, calcium gluconate and calcium sulfate.

40. according to the method for claim 39, wherein said calcium salt is a calcium acetate.

41. according to each method in the claim 33,36 or 37, wherein said monovalent cation is selected from the group of being made up of lithium, sodium, potassium and ammonium.

42. according to the method for claim 41, wherein said monovalent cation is a sodium.

43. according to each method in the claim 33,36 or 37, wherein said monovalent cation salt is selected from the group of being made up of sodium citrate, sodium phosphate and sodium acetate.

44. according to the method for claim 43, wherein said monovalent cation salt is sodium acetate.

45. according to the method for claim 33, wherein said crystallization solution further comprises the pH buffer.

46. according to the method for claim 45, wherein said pH buffer has the pH that is selected from by the following group of forming:

(a) pH between about pH6 and about pH10;

(b) pH between about pH7 and about pH10;

(c) pH between about pH6 and about pH9; And

(d) pH between about pH7.8 and about pH8.9.

47. according to the method for claim 45, wherein said pH buffer is the buffer that is selected from the group of being made up of Tris, HEPES, acetate, phosphate, citrate, borate, imidazoles and glycine.

48. according to the method for claim 36 or 38, wherein said precipitant is nonionic micromolecule or non-ionic polyalcohol.

49. according to the method for claim 48, wherein said non-ionic polyalcohol is selected from the group of being made up of Polyethylene Glycol, polyvinyl alcohol and their mixture.

50. according to the method for claim 49, wherein said Polyethylene Glycol has the molecular weight that is selected from by the following group of forming:

(a) molecular weight between about 200 and about 8000;

(b) about 6000 molecular weight;

(c) about 4000 molecular weight; And

(d) about 3350 molecular weight.

51. according to the method for claim 50, wherein said Polyethylene Glycol with about 0.5% and about 20% (w/v) between concentration be present in the described crystallization solution.

52. according to the method for claim 36 or 38, wherein said precipitant is selected from the group of being made up of aminoacid, peptide, polyamino acid and their mixture.

53. according to claim 33,36 or 37 method, wherein said human growth hormone or human growth hormone's derivant are present in the described crystallization solution with the concentration that is selected from by the following group of forming:

(a) about 1mg/ml and about 1, the concentration between the 000mg/ml;

(b) concentration between about 2mg/ml and the about 50mg/ml; And

(c) concentration between about 10mg/ml and the about 25mg/ml.

54. according to claim 33,36 or 37 method, wherein said calcium salt or described monovalent cation salt are present in the described crystallization solution with the concentration that is selected from by the following group of forming:

(a) about 0.01 and about 1M between concentration; And

(b) about 25 and about 205mM between concentration.

55. according to claim 33,36 or 37 method, wherein cultivate described crystallization solution, time and temperature are selected from the group of being made up of following:

(a) between about 0.25 day peace treaty under about 33 ℃ temperature two days;

(b) between about 0.25 day peace treaty under about 25 ℃ temperature two days; And

(c) between about 0.25 day peace treaty under about 15 ℃ temperature two days.

56. according to the method for claim 36 or 37, wherein said ion-type micromolecule is selected from the group of being made up of aminoacid, peptide and their mixture.

57. according to the method for claim 36 or 37, wherein said Ionomer be selected from by protamine, polysaccharide, polyamino acid, poly arginine, polylysine, polyglutamic acid, dendrimers,

The group that poly ornithine, polymine, chitosan and their mixture are formed.

58. according to the method for claim 57, wherein said Ionomer is protamine or poly arginine.

59. method according to claim 36 or 37, wherein, in the step (e) of claim 36 or in the step (e) of claim 37, under the temperature between about 4 ℃ and about 40 ℃, cultivate described crystallization solution about 4 to about 48 hours, till the calcium crystal or monovalent cation crystal that form human growth hormone or human growth hormone's derivant.

60. method according to claim 36 or 37, wherein, in the step (e) of the step (e) of claim 36 or claim 37, described human growth hormone or human growth hormone's derivant are present in the described crystallization solution with the concentration between about 2mg/ml and the about 100mg/ml.

61. method according to claim 36 or 37, wherein, in the step (e) of the step (e) of claim 36 or claim 37, described human growth hormone or human growth hormone's derivant are present in the described crystallization solution with the concentration between about 14.5mg/ml and the about 15.5mg/ml.

62. according to the method for claim 36 or 37, wherein said crystallization buffer is selected from the group of being made up of Tris-HCl buffer, glycine buffer, HEPES buffer, imidazole buffer, Bis-Tris buffer, AMP, AMPD, AMPSO, N-two (ethoxy) glycine, ethanolamine, glyclglycine, TAPS, taurine, Triane and their mixture.

63. according to the method for claim 36 or 37, wherein, in the step (a) of claim 36 or in the step (a) of claim 37, described crystallization buffer is present in the described crystallization solution with the concentration between about 10mM and the about 800mM.

64. according to the method for claim 36 or 37, wherein, in step (a), described crystallization buffer has the pH that is selected from by the following group of forming:

(a) pH between about 3 and about 10;

(b) pH between 6 and about 9; And

(c) pH between about 7.5 and about 10.

65. according to the method for claim 36 or 37, wherein, in the step (e) of claim 36 or in the step (e) of claim 37, the described crystallization buffer in described crystallization solution makes described solution reach the pH that is selected from by the following group of forming:

(a) pH between about 3 and about 10;

(b) pH between about 6 and about 9.5; And

(c) pH between about 7.5 and about 9.5.

66. according to the method for claim 64 or 65, wherein said buffer is selected from the group of being made up of Tris, HEPES, acetate, phosphate, citrate, borate, imidazoles and glycine.

67. according to the method for claim 40, wherein said calcium acetate is the form of aqueous solution, this aqueous solution has the pH that is selected from by the following group of forming:

(a) pH between about 3.0 and about 9.0; And

(b) pH between 7.0 and about 8.6.

68. according to the method for claim 40, wherein, in the step (e) of claim 36 or in the step (e) of claim 37, described calcium acetate is present in the described crystallization solution with the concentration that is selected from by the following group of forming:

(a) concentration between about 0.1mM and the about 205mM; And

(b) concentration between about 85mM and the about 100mM.

69. according to the method for claim 44, wherein said calcium acetate is the form of aqueous solution, this aqueous solution has the pH that is selected from by the following group of forming:

(a) pH between about 3.0 and about 9.0; And

(b) pH between 7.0 and about 8.6.

70. according to the method for claim 45, wherein, in the step (e) of claim 36 or in the step (e) of claim 37, described calcium acetate is present in the described solution with the concentration that is selected from by the following group of forming:

(a) concentration between about 0.5mM and the about 800mM; And

(b) concentration between about 100mM and the about 500mM.

71., wherein, in the step (e) of claim 36 or in the step (e) of claim 37, under the temperature between about 4 ℃ and about 37 ℃, cultivated described solution about one day to about two days according to the method for claim 36 or 37.