HK1141813A

HK1141813A - Human growth hormone crystals and methods for preparing them

Info

Publication number: HK1141813A
Application number: HK10108331.9A
Authority: HK
Inventors: C‧戈夫达恩; N‧卡拉夫; B‧P‧西米恩
Original assignee: 阿尔特斯制药公司
Priority date: 2002-12-31
Filing date: 2010-09-02
Publication date: 2010-11-19

Description

Human growth hormone crystals and method for preparing same

The present application is a divisional application of a patent application having application No. 200380109408.2, filed on 12/31/2003 entitled "human growth hormone crystals and methods for preparing them".

Technical Field

The present invention relates to crystals of human growth hormone or a human growth hormone derivative and compositions or formulations comprising them. In addition, the present invention provides a method for producing a crystal of human growth hormone or a human growth hormone derivative. The crystals of the invention are particularly useful in the treatment of mammals having disorders associated with human growth hormone deficiency or ameliorated by treatment with human growth hormone.

Background

Somatotropin or growth hormone ("GH") is a mammalian protein that contains a class of hormones synthesized and secreted in the brain by the major glands of the endocrine system, the adenohypophysis. GH and other hormones secreted by the adenohypophysis regulate the activity of cells in other endocrine glands and tissues throughout the body. In particular, GH is secreted from somatotrophic hormone cells of the adenohypophysis gland, and has a function of stimulating the synthesis and secretion of IGF-1 by the liver and other tissues, IGF-1 being a protein that controls cell division, regulates metabolic processes and exists in a free state or binds to one of 6 other proteins designated IGFBP-1 to 6. The secretion process itself is regulated by the antagonistic action of somatostatin (promoting GH release) and somatostatin (inhibiting GH release).

Human growth hormone ("hGH") is of particular interest because it functions as an important hormone in the regulation of cell and organ growth and physiological function in various stages of aging. For example, excessive production of hGH leads to gigantism in children and acromegaly in adults, whereas insufficient production of hGH leads to dwarfism in children [ Mauras et al, J.Clin.Endocrinologyand Metabolism, 85 (10); 3653-3660 (2000); frindik et al, Hormoneresearch, 51(1), 15-19 (1999); leger et al, j.clin.endocrinology and metabolism, 83(10), 3512-; pasquinono et al, Hormoneresearch, 46(6), 269-238 (1996) and chronic renal insufficiency [ Carroll et al, Trends in Endocrinology and Metabolism, 11(6), 231-238 (2000); ueland et al, J.Clin.Endocrinology and Metabolim, 87(6), 2760. 2763 (2002); simpson et al, Growth Hormone & IGF Research, 12, 1-33(2002) ]. In adults, the deficiency of hGH has an effect on the metabolic processes of proteins, carbohydrates, lipids, minerals and connective tissue and can lead to atrophy of muscle, bone or skin [ Mehlsand Haas, Growth Hormone & IGF Research, Supplement B, S31-S37 (2000); fine et al, J.Pediatrics, 136(3), 376-382 (2000); motoyama et al, Clin. exp. nephrology, 2(2), 162-165(1998) ]. Other hGH deficiencies characterized by insufficient growth include AIDS wasting syndrome [ Hirschfeld, Hormoneresearch, 46, 215-; tritos et al, am.J. medicine, 105(1), 44-57 (1998); mullgan et al, J.Parential and Enteral Nutrition, 23(6), S202-S209 (1999); torres and Cadman, Biodrugs, 14(2), 83-91(2000) and Prader-Welch syndrome [ Ritzen, Hormon Research, 56(5-6), 208 (2002); eiholzer et al, Eur. J. Pediatrics, 157(5), 368-377(1998) ].

To date, the treatment regimens lacking human hGH have mainly focused on subcutaneous injection of purified hGH produced by recombinant DNA technology. Such therapeutic agents are packaged as solutions in cartridges or as lyophilized powders that require reconstitution at the time of use. The frequency of injection varies depending on the disease to be treated and the commercially available product used. For example, dwarfism is treated by daily subcutaneous injection of recombinant hGH.

The use of subcutaneous administration as a rapid delivery route for hGH is essential for the inherent instability of the protein in solution. This instability results from the cleavage of critical intramolecular cross-links at specific positions within the amino acid sequence of the protein, which in turn disrupts the essential three-dimensional structures in the patient that are recognized by and associated with the cell surface. The cleavage or degradation mechanism of hGH is mainly coordinated by oxidation of methionine residues or deamidation of aspartic acid residues upon solubilization, thus inactivating the protein. Due to this fragility, there is a need in the art for hGH compositions or formulations that are stable and long acting, and that can be administered not only subcutaneously but also by other conventional dosage routes, such as oral, dermal and intravenous routes.

Many Commercially available hGH products have been developed in an attempt to address this need. For example, NutropinIs a suspension of injectable recombinant human growth hormone (rhGH) implanted with polylactide-co-glycolide (PLG) microspheres (see http:// www.gene.com). In addition to rhGH and PLG, the microspheres also contain components of zinc acetate and zinc carbonate. Prior to administration, the solid substance must be reconstituted with an aqueous solution comprising sodium carboxymethyl cellulose salt, polysorbate, sodium chloride and water. This suspension, which consists essentially of a polymer, is administered once or twice a month and requires injection with a 21 gauge needle. Due to the size of the microspheres and the viscosity of the product, adverse injection site reactions can occur, leading to nodules, erythema, pain, swelling, itching, fat atrophy and swelling (see http:// www.genentech.com/gene/products/information/opterustic/nucropin-depot/index.

Another hGH product that has been under development but subsequently discontinued is Albutropin^TMA genetically engineered long acting fusion protein of human albumin and human growth hormone (see http:// www.hgsi.com/products/albutin. html.) is said to exhibit an extended half-life in circulation, which is increased by about 50% over soluble native hGH. Albutropin^TMTypically administered by injection on a weekly basis and stimulates IGF-1 levels long after being cleared from the body. The biological effect of this product is similar to currently available growth hormone therapies.

Another product that has been developed is Infitropin CR^TMAn hGH formulation consisting of a polyethylene glycol-conjugated hGH molecule. This conjugated hGH requires injections once a week and is said to be released at a continuous rate with no significant burst effect [ Ross et al, J.biol.chem., 271(36), 21696-]. However, this product has been stopped.

Us patents 5,981,485 and 6,448,225 mention aqueous formulations of hGH which are said to require no reconstitution step and are administered by daily injection. Such formulations typically comprise hGH, a buffer, a non-ionic surfactant and optionally a neutral salt, mannitol, or a preservative.

A wide variety of other drug delivery techniques, such as hydrogels [ Katakam et al, J.controlled Release, 49(1), 21-26(1997) ], liposomes, oil emulsions, and biodegradable polymer microspheres, have been used in an attempt to provide sustained Release of hGH. However, the resulting formulations exhibit a burst of drug release, use harsh conditions and some of them are complicated to manufacture. These are especially true for hGH formulations based on DL-lactic acid-co-glycolic acid (PLGA) microsphere technology, since the processes for producing microspheres tend to use conditions such as elevated temperature, surfactants, organic solvents, and water/organic solvent interfaces, all of which can cause protein denaturation [ Herberger et al, Proc. Intl. Symp. controlled Release of Bioactive Materials, 23, 835-836 (1996); kim et al, Intl.J. pharmaceuticals, 229(1-2), 107-116(2001) ].

Some of the formulations described above require that the hGH be stored in a lyophilized state, which can be a time consuming and expensive process. U.S. Pat. Nos. 5,780,599 and 6,117,984 relate to divalent cation crystallization of hGH and methods of producing divalent cation crystallization of hGH without the need for a lyophilization step.

Despite efforts to address the disadvantages of conventional hGH products, including instability upon storage and injection, short in vivo half-life, burst effects, lack of oral bioavailability, and difficulty and frequency of administration, there remains a need for improved hGH formulations. To meet this need, the present invention advantageously provides crystals of human growth hormone producing stable, long acting hGH.

Disclosure of Invention

The present invention relates to stable, long acting, convenient and patient friendly crystals of human growth hormone or a human growth hormone derivative. The present invention further provides a composition of crystals of human growth hormone or a human growth hormone derivative, including pharmaceutically acceptable compositions thereof. The invention further provides methods of making such crystals, and compositions comprising them. The crystals and compositions of the invention are advantageously used in methods of treating individuals having disorders associated with human growth hormone deficiency or ameliorated by treatment with human growth hormone.

Crystals of human growth hormone or human growth hormone derivatives, or compositions or formulations containing them, have several advantages, including: capable of once weekly administration in the form of a ready-to-use crystalline suspension, safety, efficacy, purity, stability, re-suspendability and injectability over a short period of time. In view of the present disclosure, those skilled in the art will appreciate further objects of the invention, including improvements in hGH crystals and compositions or formulations comprising them, as compared to conventional hGH formulations.

Detailed description of the invention

Definition of

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include singular terms. In general, the specialists and techniques associated with column chromatography, light microscopy, UV-VIS spectroscopy, pharmacokinetic analysis, recombinant DNA methods, peptide and protein chemistry, nucleic acid chemistry and molecular biology described herein are well known and commonly used in the art.

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

the term "Growth Hormone (GH)" generally refers to a growth hormone secreted by the pituitary gland of mammals. Although not an exhaustive list, examples of mammals include humans, apes, monkeys, rats, pigs, dogs, rabbits, cats, cows, horses, mice, rats, and goats. According to a preferred embodiment of the invention, the mammal is a human.

"human growth hormone (hGH)" means a protein having the amino acid sequence, structural and functional characteristics of natural human growth hormone. As used herein, human growth hormone (hGH) also includes any isoform of native human growth hormone, including, but not limited to, isoforms of 5, 17, 20, 22, 24, 36 and 45kDa in molecular mass [ Haro et al, j.chromatography B, 720, 39-47(1998) ]. Thus, the term hGH includes 191 amino acid sequences of natural hGH (somatotropin) and 192 amino acid sequences containing the N-terminal methionine (Met-hGH) and human methionine growth hormone [ U.S. Pat. Nos. 4,342,832 and 5,633,352 ]. hGH may also be obtained by isolation and purification from biological sources or by recombinant DNA methods. If prepared by recombinant DNA methods, hGH is designated recombinant human growth hormone (rhGH). Met-hGH is typically prepared by recombinant DNA methods.

The term "human growth hormone derivative" refers to a protein having an amino acid sequence comparable to that of naturally occurring human growth hormone. The term "comparable" refers to an amino acid sequence that is between 2% and 100% homologous to the 191 amino acid sequences of hGH or 192 amino acid sequences of Met-hGH. In various embodiments of the invention, the human growth hormone derivative comprises organic cations of hGH or Met-hGH, substitution, deletion and insertion variants of biosynthetic hGH or Met-hGH proteins, post-translationally modified hGH or Met-hGH proteins, including deamidation, phosphorylation, glycosylation (glycosylation), acetylation, polymerization and enzymatic cleavage reactions [ Haro et al, J.Chromatogray B, 720, 39-47(1998) ], chemically modified hGH or Met-hGH proteins from biological origin, polypeptide analogs comprising amino acid sequences similar to hGH or Met-hGH, and chemically synthesized peptides.

Methods for preparing hGH or Met-hGH include isolation from biological sources, recombinant DNA methods, synthetic chemical routes, or combinations thereof. To date, genes encoding different DNA sequences of hGH include hGH-N and hGH-V [ Haro et al, J.Chromatology B, 720, 39-47 (1998); Bennani-Baiti et al, Genomics, 29, 647-652(1995) ].

The term "valence" is defined as the ability of an element to bind to other elements, which is determined by the number of electrons in the outermost shell of the atom and is expressed as the number of atoms of hydrogen (or any other standard monovalent element) that can bind to (or replace) its atom [ Webster's New world dictionary of Science, Lindley, D.and Moore T.H., eds., Macmillan, New York, New York, 1998 ] A.]. The terms "monovalent cation" and "divalent cation" refer to an ion having a positive charge in a monovalent state or a divalent state, respectively. Cations with different valence states can be organic or inorganic in nature. Examples of monovalent inorganic cations include ammonium (NH)⁴⁺) And elements of periodic table group I (H)⁺、Li⁺、Na⁺、K⁺、Rb⁺、Cs⁺And Fr⁺) And the divalent inorganic cation comprises a group II element (Be)⁺、Mg²⁺、Ca²⁺、Sr²⁺、Ba²⁺、Mn²⁺、Co²⁺、Ni²⁺、Cu²⁺、Zn²⁺、Cd²⁺、M0²⁺And Ra²⁺)。

"calcium crystals of human growth hormone or a human growth hormone derivative" refers to human growth hormone or its derivative which has been crystallized in the presence of divalent calcium ions. Divalent calcium ions are introduced into the crystallization solution in the form of calcium salts. In a preferred embodiment, the calcium crystals of human growth hormone or human growth hormone derivative comprise from about 1 to about 500 calcium molecules per monomer or monomer chain of human growth hormone or human growth hormone derivative. In a more preferred embodiment, the calcium crystals of human growth hormone or human growth hormone derivative comprise from about 1 to about 140 calcium molecules per monomer or monomer chain of human growth hormone or human growth hormone derivative.

The term "calcium salt" includes inorganic and organic counter ions or molecules that form ionic bonds with calcium ions. Examples of different calcium salts include calcium acetate hydrate, calcium acetate monohydrate, calcium acetylacetonate hydrate, calcium L-ascorbate dihydrate, calcium bis (6, 6, 7, 7, 8, 8, 8-heptafluoro-2, 2-dimethyl-3, 5-octanedionate), calcium bis (2, 2, 6, 6-tetramethyl-3, 5-heptanedionate), calcium bromide, calcium carbonate, calcium chloride dihydrate, calcium chloride hexahydrate, calcium chloride hydrate, calcium citrate tetrahydrate, calcium dihydrogen phosphate, calcium 2-ethylhexanoate, calcium fluoride, calcium gluconate, calcium hydroxide, calcium hypochlorite, calcium iodate, calcium iodide hydrate, solid electrolyte I calcium, calcium molybdate, calcium nitrate, calcium oxalate hydrate, calcium oxide, calcium pantothenate, calcium propionate, calcium pyrophosphate and calcium sulfate. In a preferred embodiment of the invention, the calcium salt is selected from the group consisting of calcium acetate, calcium chloride, calcium sulfate and calcium gluconate. In a more preferred embodiment, the calcium salt is calcium acetate.

"organic cationic crystal of human growth hormone or human growth hormone derivative" refers to human growth hormone which has been crystallized in the presence of an organic cation. The term "organic cation" refers to a positively charged atom or group of atoms comprising carbon. Examples of organic cations include quaternary ammonium cations, Tetraethylammonium (TEA), tributylmethylammonium (TBuMA), procainamide ethobromide (paeb), azidoprocainamide N-methyliodide (APM), d-tubocurarine, difocurarine bromide (metocuronium), rocuronium bromide, 1-methyl-4-phenylpyridine, choline and N- (4, 4-axo-N-pentyl) -21-deoxyamalinium (APDA).

hGH is commercially available in lyophilized form and is typically produced by recombinant DNA methods. According to the present invention, crystallization of hGH is generally accomplished by preparing a buffered solution of hGH, purifying and/or desalting, dialyzing and concentrating the solution and adding a monovalent or divalent cation or salt to the solution. The latter step results in the formation of bound hGH organic or inorganic cations.

A preferred embodiment of the present invention relates to monovalent cation crystals of hGH or hGH derivatives. In a more preferred embodiment, the monovalent cation is selected from the group consisting of lithium, sodium, potassium and ammonium. In a most preferred embodiment, the monovalent cation is sodium. In a most preferred embodiment, the human growth hormone or human growth hormone derivative comprises from about 1 to about 500 monovalent cation molecules per monomer or monomer chain of human growth hormone or human growth hormone derivative.

The term "monovalent cation salt" includes inorganic and organic counterions or molecules that form ionic bonds with monovalent ions. In a preferred embodiment, the monovalent cation salt is a sodium salt. In a more preferred embodiment, the sodium salt is selected from the group consisting of sodium citrate, sodium phosphate and sodium acetate. In a most preferred embodiment, the sodium salt is sodium acetate.

Another preferred embodiment of the present invention relates to protamine crystals of hGH or hGH derivatives. Likewise, in yet another preferred embodiment, the present invention relates to poly-arginine (polyarginine) crystals of hGH or hGH derivative.

Another preferred embodiment of the present invention comprises monovalent or divalent crystals of hGH or hGH derivatives bound to or co-crystallized with protamine or polyarginine. More preferably, the crystals are sodium crystals complexed or co-crystallized with protamine or polyarginine.

The soluble form of hGH can be characterized by a variety of methods, including reverse phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography high performance liquid chromatography (SEC-HPLC), and Hydrophobic Interaction Chromatography (HIC) [ Wu et al, J.chromatograpy, 500, 595-606 (1990); "Hormones Drugs", FDA publication (1982) ]. On the other hand, crystalline forms of hGH may be characterized by optical microscopy and X-ray diffraction. In general, the crystallization conditions will determine the shape of the protein crystal, i.e., a shape selected from the group consisting of spherical, acicular, rod-like, disk-like (hexagonal and square), rhombohedral, cubic, bipyramidal, and prismatic.

According to the present invention, crystals of hGH or hGH derivative form a rod-like or needle-like morphology when imaged by optical microscopy. In one embodiment, the crystals of hGH or hGH derivative form rods or needles with a length of between about 0.1 and about 200 μm. In a preferred embodiment, the crystals of hGH or hGH derivative form rods or needles with a length of between about 3 and about 100 μm. In a more preferred embodiment, the crystals of hGH or hGH derivative form rods or needles with a length of between about 10 and about 25 μm.

Another embodiment of the invention relates to a composition comprising calcium of hGH or hGH derivative, a monovalent cation, protamine or poly-arginine crystals and a pharmaceutically acceptable excipient. In yet another preferred embodiment, crystals of hGH or hGH derivative and excipient are present in such a composition in a molar ratio of hGH to excipient of from about 1: 250 to about 1: 20. In another alternative preferred embodiment, the crystals of hGH or hGH derivative and excipient are present in a molar ratio of hGH to excipient of about 3: 1 to about 1: 10. In yet another preferred embodiment, the crystals of hGH or hGH derivative and excipient are present in a molar ratio of hGH to excipient of about 1: 10 to about 1: 0.125. In a preferred embodiment, crystals of hGH or hGH derivatives are grown with sodium acetate, which may be crystallized or coated with polyarginine or protamine.

The crystals of human growth hormone or human growth hormone derivative may be combined with any pharmaceutically acceptable excipient. According to the present invention, a "pharmaceutically acceptable excipient" is an excipient used in a pharmaceutical composition to function as a filler or filler composition. Preferred excipients included in this category are: 1) amino acids such as glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline; 2) sugars, for example, monosaccharides such as glucose, fructose, galactose, mannose, arabinose, xylose, ribose; 3) disaccharides, such as lactose, trehalose, maltose, sucrose; 4) polysaccharides such as maltodextrin, dextran, starch, glycogen; 5) acyclic polyhydric alcohols such as mannitol, xylitol, lactitol, sorbitol; 6) glucuronic acid, galacturonic acid; 7) cyclodextrins, such as methyl cyclodextrin, hydroxypropyl-beta-cyclodextrin, and the like; 8) inorganic molecules such as sodium chloride, potassium chloride, magnesium chloride, sodium and potassium phosphates, boric acid, ammonium carbonate and ammonium phosphate; 9) organic molecules such as acetate, citrate, ascorbate, lactate; 10) emulsifying or solubilizing/stabilizing agents such as acacia, diethanolamine, glycerol monostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol, poloxamers, polysorbates, sodium lauryl sulfate, stearic acid, sorbitan monolaurate, sorbitan monostearate, and other sorbitan derivatives, polyoxyl derivatives, waxes, polyoxyethylene derivatives, sorbitan derivatives; and 11) viscosity increasing agents such as agar, alginic acid and its salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives, propylene carbonate, polyethylene glycol, hexylene glycol, tyloxapol. Salts of these compounds may also be used. A more preferred group of excipients includes sucrose, trehalose, lactose, sorbitol, lactitol, mannitol, inositol, sodium and potassium salts, such as acetates, phosphates, citrates and borates, glycine, arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol, methoxypolyethylene glycol, gelatin, hydroxypropyl-beta-cyclodextrin, polylysine and polyarginine.

In one embodiment of the invention, the excipient is selected from the group consisting of amino acids, salts, alcohols, carbohydrates, proteins, lipids, surfactants, polymers, polyamino acids and mixtures thereof. In a preferred embodiment, the excipient is selected from the group consisting of protamine, polyvinyl alcohol, cyclodextrin, dextran, calcium gluconate, polyamino acids, such as polyarginine, polylysine and polyglutamic acid, polyethylene glycol, dendrimers, polyornithine (polyornithine), polyethyleneimine, chitosan and mixtures thereof. In a more preferred embodiment, the excipient is selected from the group consisting of protamine, polyarginine, polyethylene glycol and mixtures thereof.

According to the present invention, the crystals of human growth hormone or human growth hormone derivative may also be combined with a carrier or excipient which is a substance which, when added to a therapeutic agent, accelerates or improves the action of the therapeutic agent. [ The On-Line medical dictionary, http:// cancerweb. nc.ac.uk/omd/index. html ]. Examples of carriers or excipients include, for example, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium, trisilicates, cellulose-based substances and polyethylene glycols. Carriers or excipients in the form of gel matrices may include, for example, sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene block copolymers, polyethylene glycol and wood wax alcohols (wood wax alcohols).

In yet a more preferred embodiment, the excipient is protamine. Furthermore, crystals of hGH or hGH derivative and protamine are present in a ratio of hGH to protamine of about 5: 1 to about 1: 10 (w/w). This ratio may also 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 an alternative 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). Moreover, in a further embodiment, this ratio is about 5: 1 (w/w).

In another aspect of the invention, the pharmaceutically acceptable excipient is selected from the group consisting of polyamino acids, including polylysine, polyarginine, and polyglutamic acid. In a preferred embodiment of the invention, the excipient is polylysine. In a more preferred embodiment, the polylysine has a molecular weight of between about 1,500 and about 8,000 kD. In another embodiment, the crystals of hGH or hGH derivative and polylysine are present in a ratio (w/w) of hGH to polylysine of about 5: 1 to about 40: 1. This ratio may also 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 an alternative embodiment, this ratio is between about 5: 1 to about 1: 50 (w/w). Moreover, in a further embodiment, this ratio is about 5: 1 (w/w).

In yet another preferred embodiment of the invention, the excipient is polyarginine. In a more preferred embodiment, the polyarginine has a molecular weight between about 15,000 and about 60,000 kD. In another embodiment, the crystals of hGH or hGH derivative and polyarginine are present in a ratio of hGH to polyarginine of about 5: 1 to about 40: 1 (w/w). This ratio may also 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 an alternative embodiment, this ratio is between about 5: 1 to about 1: 50 (w/w). In another embodiment, this ratio is in the range of about 12: 1 to about 15: 1 (w/w). Moreover, in a further embodiment, this ratio is about 5: 1 (w/w).

An embodiment according to the invention comprises an injectable crystalline suspension comprising crystals of hGH or hGH derivative in an amount of about 20 mg/ml. This suspension is characterized by easy resuspension, slow sedimentation and a time action of about 7 days. It can be injected once a week with a 30 gauge syringe and provide 80% level payload. The suspension was pure as reflected by the parameters 0.02% aggregation (SE-HPLC) and 2.3% of the protein of interest (RP-HPLC). This purity is maintained under refrigerated conditions for at least 4 months.

One embodiment of the present invention relates to crystals of hGH or hGH derivatives which are characterized by a dissolution-delaying behavior when placed in a subject, as compared to conventional soluble hGH or hGH formulations. According to the present invention, the dissolution characteristics of crystals of hGH or hGH derivatives are dissolution parameters in vivo or in vitro.For example, in vitro dissolution is described as the concentration of soluble hGH (expressed as the percentage of total hGH or hGH derivative crystals initially present or mg of total hGH or hGH derivative crystals) obtained every 15 minutes or every washing step in one continuous dissolution process (see example 5). In one embodiment of the invention, crystals of hGH or hGH derivative are characterized by exposure to a dissolution buffer (50mM HEPES (pH7.2), 140mM NaCl, 10mM KC1 and 0.02% (v/v) NaN at a temperature of 37 deg.C₃) In particular, about 2 to about 16% of the in vitro dissolution rate of said crystals per washing step, wherein the concentration of hGH or hGH derivative in the solution is present at about 2 mg/ml. In another embodiment, the crystals of hGH or hGH derivative are characterized by an in vitro dissolution rate of about 0.04 to about 0.32mg of said crystals per washing step in a continuous dissolution process (see example 5). On the other hand, in vivo dissolution is described by serum levels of hGH in the mammal over time after a single injection of hGH into the mammal.

In mammals, GH stimulates tissues to synthesize and secrete IGF-1, a protein that in turn plays a role in cell division and metabolism. As will be appreciated by those skilled in the art, serum hGH and IGF-1 levels depend on a number of factors, including physiological and therapeutic related factors. Such factors include, but are not limited to: physiological factors, such as: birth and bone age, sex, body weight, developmental stage (e.g., increased levels at puberty) and treatment-related factors such as dose, rate of administration (kinetics) and route of administration. Likewise, those skilled in the art will appreciate that different levels of hGH and IGF-1 may also be beneficial for different patient populations from a safety and efficacy standpoint.

Adults or children with various hGH deficiencies, disease states or syndromes may be treated by various regimens of exogenously delivered hGH with crystals of hGH or crystals of hGH derivatives according to the present invention. For example, an endocrinologist may begin treatment of a child at a dose of about 0.2 mg/kg/week, increasing the dose to 0.3 mg/kg/week over a period of weeks or months, and around puberty, the dose may further increase to about 0.7 mg/kg/week. As will be appreciated by those skilled in the art, the level of such exogenous delivery of hGH to an adult or child in need of hGH delivery also depends on the physiological level or concentration at which the hGH is present.

The hGH dosage regimen for adults and children is expressed in mg/kg or international units (IU/kg). Such regimens are typically scheduled on a daily or weekly basis, i.e., mg/kg/day or mg/kg/week. With this in mind, according to one embodiment of the invention, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, e.g., a single administration of about 9mg per week per 30kg child, provides a serum concentration of hGH greater than about 10ng/ml in vivo on days 1 and 2 after administration, a serum concentration of hGH greater than about 5ng/ml in vivo on days 3 and 4 after administration, and a serum concentration of hGH about 0.3ng/ml in vivo on days 5 and 7 after administration. Alternatively, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, provides an in vivo hGH serum concentration in said mammal of about 0.3ng/ml to about 2,500ng/ml hGH, preferably about 0.5ng/ml to about 1,000ng/ml hGH, more preferably about 1ng/ml to about 100ng/ml hGH, for any one of about 0.5 hour to about 40 days post-administration, preferably about 0.5 hour to about 10 days, 7 days or 1 day post-administration. Likewise, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, provides an in vivo serum concentration of greater than about 2ng/ml hGH, preferably greater than about 5ng/ml hGH, more preferably greater than about 10ng/ml hGH, in said mammal for any one of about 0.5 hour to about 40 days post-administration, preferably about 10 days, 7 days or 1 day post-administration. In a more preferred embodiment of the invention, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, provides an in vivo serum concentration of hGH greater than about 0.3ng/ml in said mammal for any one of a period of time from about 0.5 hour after administration to about 40 days, preferably 10 days, 7 days or 1 day after administration. According to one embodiment of the invention, a single administration of crystals of hGH or hGH derivative, or a composition comprising these crystals, provides a serum concentration of hGH greater than about 10ng/ml in vivo on days 1 and 2 after administration, a serum concentration of hGH greater than about 5ng/ml in vivo on days 3 and 4 after administration, and a serum concentration of hGH greater than about 0.3ng/ml in vivo on days 5 and 7 after administration. Moreover, in a further embodiment, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, provides an in vivo serum concentration of greater than about 0.3ng/ml hGH, for about 0.5 hour to about 10 days after administration.

According to one embodiment of the invention, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, provides an in vivo IGF-1 serum elevation above the baseline IGF-1 level prior to said administration, greater than about 50ng/ml from about 10 hours to about 72 hours after administration, and between about 0.5ng/ml to about 50ng/ml from about 72 hours to about 15 days after administration, preferably about 10 days after administration. Alternatively, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, provides an in vivo IGF-1 serum elevation of about 5ng/ml to about 2,500ng/ml, preferably about 100ng/ml to about 1,000ng/ml, for about 0.5 hour to about 40 days after administration, preferably about 7 days after administration. Alternatively, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, may provide an in vivo IGF-1 serum elevation of more than about 50ng/ml, preferably more than about 100ng/ml, according to the invention, for about 0.5 hour to about 40 days, preferably about 7 days, after administration. According to one embodiment of the invention, a single administration of crystals of hGH or hGH derivative, or a composition comprising such crystals, provides an in vivo IGF-1 serum elevation above the baseline IGF-1 level prior to said administration of greater than about 50ng/ml from about 10 hours to about 72 hours after administration, and between about 0.5ng/ml to about 50ng/ml from about 72 hours to about 15 days after administration or from about 72 hours to about 10 days after administration.

According to the present invention, a single administration is defined as between about 0.01 mg/kg/week and about 100 mg/kg/week crystals of hGH or hGH derivative, or a composition comprising such crystals, wherein the volume administered is between 0.1ml and about 1.5 ml. For example, a growth hormone deficiency in a child may be administered at about 0.3 mg/kg/week, e.g. about 9mg for a 30kg child, with crystals of hGH or hGH derivative, or a composition comprising such crystals. Turner's syndrome may be administered at about 0.375 mg/kg/week, e.g. about 11.25mg for a 30kg child, with crystals of hGH or hGH derivative, or a composition comprising such crystals. Alternatively, hGH may be administered as a growth hormone deficiency in adults at about 0.2 mg/kg/week, for example about 16mg for an 80kg adult. AIDS wasting disease may be administered hGH at 6 mg/day, e.g. 42 mg/week.

In yet another embodiment of the present invention, crystals of hGH or hGH derivative, or a composition comprising such crystals, exhibit relative bioavailability in mammals similar to that of soluble hGH. The crystals according to the invention have a relative bioavailability of at least 50% or more compared to the relative bioavailability of soluble hGH delivered by the same route, wherein said bioavailability is determined by the area under the curve (AUC) of the total in vivo serum concentration of hGH for said soluble hGH and said crystals. Crystals of hGH or hGH derivatives are therefore characterized by an advantageous rate of dissolution in vivo.

The present invention further provides a method of administering crystals of hGH or hGH derivative to a mammal having a disorder associated with human growth hormone deficiency or ameliorated by treatment with hGH. The method comprises the step of administering to the mammal a therapeutically effective amount of crystals of hGH or hGH derivative. Alternatively, the method comprises the step of administering to the mammal a therapeutically effective amount of a composition comprising crystals of hGH or hGH derivative, alone or with excipients. Different embodiments of the crystals of hGH or hGH derivatives according to the invention are: calcium crystals, monovalent crystals, protamine crystals or poly-arginine crystals of hGH or hGH derivatives. Such crystals, or compositions containing them, may be administered on a time schedule of about once every three days, about once a week, about once every two weeks, or about once a month.

Disorders associated with hGH deficiency that may be treated according to the present invention include, but are not limited to: adult growth hormone deficiency, childhood growth hormone deficiency, prader-willi syndrome, turner's syndrome, short bowel syndrome, chronic renal insufficiency, idiopathic short stature, dwarfism, hypopituitarism, bone regeneration, female infertility, intrauterine growth retardation, AIDS-related cachexia, crohn's disease, burns, and other genetic and metabolic disorders. In one embodiment of the invention, the disorder is a childhood growth hormone deficiency and in children undergoing treatment, the treatment results in an annual growth rate of between about 7cm and about 11 cm.

In another embodiment of the invention, calcium crystals of hGH or hGH derivatives may act as a useful adjunct in the treatment of bone, as well as in the treatment of human growth hormone deficiency in mammals.

The present invention also provides a method for inducing weight gain in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of crystals of hGH or hGH derivative. Alternatively, such methods comprise the step of administering to said mammal a therapeutically effective amount of a composition comprising crystals of hGH or hGH derivative and an excipient. In one embodiment of such methods, the body weight gain induced in hypophysectomized rats is between about 5% and about 40% after administration of the crystals by injection once a week.

Crystals of hGH, crystals of hGH derivatives or compositions comprising them alone, or with excipients, may be administered alone or as part of a pharmaceutical, therapeutic or prophylactic formulation. They may be administered by any conventional route of administration including, for example, parenteral, oral, pulmonary, nasal, otic, anal, dermal, ocular, intravenous, intramuscular, intraarterial, intraperitoneal, mucosal, sublingual, subcutaneous, transdermal, topical, buccal or intracranial routes.

In one embodiment of the invention, the crystals of hGH or hGH derivative, or a composition comprising them, are administered by oral or parenteral route, with or without excipients. In a preferred embodiment, the crystals of hGH or hGH derivative, or a composition comprising them, are administered by subcutaneous or intramuscular route, with or without excipients.

In a preferred embodiment, the crystals or compositions of the invention are administered by subcutaneous route using a needle having a gauge greater than or equal to 27. In one embodiment of the invention, the needle gauge may be equal to 30. The crystals or compositions may be administered from a pre-filled syringe or a variable dose infusion pump. Alternatively, they are administered by needle-free injection.

The present invention advantageously allows for the sustained release of hGH into mammals. In one embodiment, the crystal or composition according to the invention is administered about once a week. In another embodiment, the crystal or composition according to the invention is administered about once every two weeks. In yet another embodiment, the crystal or composition according to the invention is administered about once a month. Those skilled in the art will appreciate that the specific treatment regimen will depend upon factors such as the condition to be treated, the age and weight of the patient to be treated, the general physical condition of the patient, and the judgment of the treating physician.

According to one embodiment, a composition comprising crystals of hGH or hGH derivative of the invention is characterized by a concentration of hGH greater than about 0.1 mg/ml. For example, the concentration may be between about 0.1mg/ml and about 100 mg/ml. Alternatively, those compositions may be characterized by hGH concentrations of between about 1mg/ml and about 100mg/ml or between about 10mg/ml and about 100 mg/ml. Such compositions also include the following components: mannitol-from about 0.5mg/ml to about 100 mg/ml; sodium acetate-about 5mM to about 250mM (preferably about 25mM to about 150 mM); tris HCl-about 5mM to about 100 mM; a pH of about 6.0 to about 9.0 (preferably about 6.5 to about 8.5); PEG (MW 800-8000, preferably 3350, 4000, 6000 or 8000) -0 to about 25%; protamine, preferably hGH: protamine in a 3: 1 ratio; and polyarginine, preferably hGH polyarginine in a 5: 1 ratio. Such compositions may optionally comprise: sucrose-0 mg/ml to about 100 mg/ml; amino acids (e.g., arginine and glycine) -0mg/ml to about 50 mg/ml; preservatives (antimicrobials, phenol, m-cresol (matacresol), benzyl alcohol, parabens) -0% to about 5% (preferably 0% to about 0.9%); and polysorbate-0 mg/ml to about 10 mg/ml. According to one embodiment, the composition according to the invention is characterized by an 80% payload.

A preferred formulation vehicle according to the invention comprises about 100mM sodium acetate, about 5% PEG6000MW and about 25mM Tris HCl, pH 7.5. hGH compositions prepared using such carriers may comprise: about 9.35mg/ml crystalline hGH and about 1.81mg/ml polyarginine (or about 3.12mg/ml protamine). As will be appreciated by those skilled in the art, given that a composition according to the present invention may comprise an hGH concentration of about 1mg/ml to about 100mg/ml, the concentration of polyarginine (or protamine) should be adjusted so as to be sufficient to maintain a 5: 1 hGH: polyarginine (w/w) ratio or a 3: 1 hGH: protamine (w/w) ratio and to maintain a low solubility and release of hGH of about 5 ng/ml. For example, for the formulations described above, if the desired hGH concentration is about 20mg/ml, the polyarginine (or protamine) concentration should be about 4 mg/ml.

The present invention further provides a method for preparing crystals of hGH or hGH derivative. One such method comprises the steps of: (a) mixing a solution of human growth hormone or a human growth hormone derivative with a crystallization solution, the crystallization solution comprising a salt and an ionic polymer; and (b) incubating the solution at a temperature between about 4 ℃ and about 37 ℃ for greater than about 12 hours until crystals of human growth hormone or a human growth hormone derivative are formed. In another embodiment, the method comprises the steps of: (a) mixing a solution of human growth hormone or a human growth hormone derivative with a crystallization solution, the crystallization solution comprising a salt and a precipitating agent; and (b) incubating the solution at a temperature between about 4 ℃ and about 37 ℃ for greater than about 16 hours until crystals of human growth hormone or a human growth hormone derivative are formed. In another embodiment, the solution in step (b) of any of the above methods may be incubated at a temperature of about 15 ℃ for greater than about one week. In a preferred embodiment, the crystals of hGH or hGH derivative are calcium crystals, monovalent cation crystals, protamine crystals or polyarginine crystals and the ionic polymer is protamine or polyarginine. In another embodiment, the ionic polymer is polylysine or polyornithine (polyornithine). In yet another embodiment, the ionic polymer is a mixture of any two or more of protamine, polyarginine and polylysine.

The salt in step (a) of the above process may be monovalent or divalent and may be inorganic or organic. A preferred embodiment of the divalent salt is a calcium salt. In a more preferred embodiment, the calcium salt is selected from the group consisting of calcium acetate, calcium chloride, calcium gluconate and calcium sulfate. In a still more preferred embodiment, the calcium salt is calcium acetate. In another preferred embodiment, the monovalent cation is selected from the group consisting of lithium, sodium, potassium and ammonium. In a more preferred embodiment, the monovalent cation is sodium.

In an alternative preferred embodiment of the invention, the monovalent cation salt is a sodium salt. In a more preferred embodiment, the sodium salt is selected from the group consisting of sodium citrate, sodium phosphate and sodium acetate. In a still more preferred embodiment, the sodium salt is sodium acetate.

In the above method, when the salt is a calcium salt or a monovalent cation salt, it is present in the crystallization solution of step (a) at a concentration of between about 0.01mM and about 1M. In a preferred embodiment, the concentration is between about 25 and about 205 mM. When the salt is a calcium salt, it is present in the crystallization solution of step (a) in a concentration of between about 0.01mM and 235 mM.

In a preferred embodiment, the crystallization solution of step (a) further comprises a pH buffer. In a more preferred embodiment, the buffer has a pH between about pH6 and about pH 10. In a more preferred embodiment, the pH of the buffer is between about pH7.5 and about pH 10. In a more preferred embodiment, the pH of the buffer is between about pH7.0 and about pH 10. In yet more preferred embodiments, the pH of the buffer is between about pH6 and about pH 9. In yet a more preferred embodiment, the pH of the buffer is between about pH7.8 and about pH 8.9.

In another aspect of the above method, the buffer in step (a) is selected from the group consisting of Tris, HEPES, acetate, phosphate, citrate, borate, imidazole and glycine. In a preferred embodiment of the above process, the buffer in step (a) is selected from the group consisting of bicarbonate, imidazole-malate, glycine, 2-bis (hydroxymethyl) -2, 2', 2 "-nitrilotriethanol (" bis-tris "), carbonate, N- (acetylamino) -iminodiacetic acid, 2-amino-2-methyl-1, 3 propanediol and (N- (1-acetylamino) -2-aminoethanesulfonic acid.

In one of the above processes, the precipitating agents used to prepare crystals of hGH or hGH derivatives are typically polymeric, comprising low molecular weight alcohols and protamine. In another embodiment of the present invention, the precipitating agent in step (a) of one of the above processes is a non-ionic polymer. In a preferred embodiment, the non-ionic polymer is selected from the group consisting of alcohols, polyethylene glycol (PEG), and polyvinyl alcohol or ethanol. In a more preferred embodiment, the precipitating agent is isopropanol or ethanol. In yet a more preferred embodiment, the PEG has a molecular weight between about 200 and about 8000. The PEG may have a molecular weight of 3350, 4000, 6000 or 8000. In a more preferred embodiment, the PEG has a molecular weight of about 6000. In yet another preferred embodiment, PEG is present at a concentration of between about 0.5% and about 12% w/v.

In another embodiment of one of the above processes, the precipitating agent in step (a) is a non-ionic polymer. In a preferred embodiment, the non-ionic polymer is selected from the group consisting of protamine, polyarginine, polyornithine and polylysine.

The mixing step (a) of the above method comprises mixing a solution of hGH or hGH derivative with a crystallization solution. In one embodiment of said method, the concentration of hGH or hGH derivative obtained in said crystallization solution is between about 1mg/ml and about 1,000 mg/ml. In a preferred embodiment, the hGH or hGH derivative in said solution is present in a concentration of between about 2mg/ml and about 50 mg/ml. In a further embodiment, the hGH or hGH derivative in said solution is present in a concentration of between about 10mg/ml and about 25 mg/ml.

In a preferred embodiment of the above-described method for preparing crystals of hGH or hGH derivative, the solution comprising hGH or hGH derivative and the crystallization solution is incubated at a temperature of about 33 ℃ for about 0.25 days to about two days in step (b). Alternatively, the temperature may be about 37 ℃. In another embodiment, the solution comprising hGH or hGH derivative and the crystallization solution is incubated at a temperature of about 25 ℃ for about 0.25 days to about two days. In yet another embodiment, the solution comprising hGH or hGH derivative and the crystallization solution is incubated at a temperature of about 15 ℃ for about 0.25 days to about two days.

The present invention further provides alternative methods of preparing crystals of hGH, crystals of hGH derivatives or compositions comprising such crystals and excipients. The method comprises the following steps: (a) mixing a solution of hGH or hGH derivative with a crystallization buffer to produce a crystallization solution; (b) adding deionized water to the crystallization solution; (c) adding an ionic small molecule or ionic polymer to the crystallization solution; (d) adding a salt to the crystallization solution; and (e) incubating the crystallization solution at a temperature of about 10 ℃ and about 40 ℃ for about 2 to about 168 hours until crystals of hGH or hGH derivative are formed. In a further embodiment of the invention, the incubation is carried out for about 4 to about 48 hours. In another preferred embodiment, the crystallization solution in step (e) of the above process is incubated at a temperature between about 4 ℃ and about 40 ℃ for about 4 to about 48 hours until crystals of hGH or hGH derivative are formed. According to an alternative embodiment, the above process is carried out with any step after step (b). The optional step comprises adding a precipitating agent to the crystallization solution. In a further embodiment of the above process, step (c) is optional. Regardless of whether any of those steps are used, in a preferred embodiment, the crystallization solution of step (e) is incubated at a temperature between about 15 ℃ and about 37 ℃ for about 1 to about 2 days. In another preferred embodiment, the crystallization solution of step (e) is incubated at a temperature between about 4 ℃ and about 37 ℃ for about 1 to about 2 days.

In a preferred embodiment according to the present invention, in step (d) of the above process, the salt is a calcium salt or a monovalent cation salt. In a preferred calcium crystal embodiment, the calcium salt is selected from the group consisting of calcium acetate, calcium chloride, calcium gluconate, and calcium sulfate. In yet a more preferred embodiment, the calcium salt is calcium acetate. In a preferred embodiment, the calcium acetate is in the form of an aqueous solution having a pH between about 3 and about 9.0. In a more preferred embodiment, the aqueous calcium acetate solution has a pH between about 7.0 and about 8.6. In another embodiment, the calcium acetate in the solution of step (e) is present at a concentration between about 0.1mM and about 205 mM. In a more preferred embodiment, the calcium acetate in the crystallization solution in step (e) is present at a concentration of between about 85mM and about 100 mM.

In a more preferred embodiment, the monovalent cation salt is selected from the group consisting of lithium, sodium, potassium and ammonium. In still another more preferred embodiment, the monovalent cation salt in step (d) of the above method is sodium. Similarly, in a more preferred embodiment, the monovalent cation salt is selected from the group consisting of sodium citrate, sodium phosphate and sodium acetate. In yet a more preferred embodiment, the monovalent cation salt is sodium acetate. In a preferred embodiment, the sodium acetate is in the form of an aqueous solution having a pH between about 3 and about 9.0. In a more preferred embodiment, the aqueous sodium acetate solution has a pH between about 7.0 and about 8.6. In another embodiment, the sodium acetate in the crystallization solution of step (e) is present at a concentration between about 0.5mM and about 800 mM. In a more preferred embodiment, the sodium acetate in the crystallization solution of step (e) is present at a concentration of between about 100mM and about 500 mM. The concentration may also be between about 85mM and about 100 mM.

In yet another preferred embodiment, the hGH or hGH derivative in the crystallization solution in step (e) of the above method is present in a concentration of between about 2mg/ml and about 17.5 mg/ml. In another preferred embodiment, the hGH or hGH derivative in the crystallization solution in step (e) of the above method is present in a concentration of between about 14.5mg/ml and about 15.5 mg/ml. In a further embodiment, the hGH or hGH derivative in the crystallization solution of step (e) is present in a concentration of between about 2mg/ml and about 100 mg/ml.

In a preferred embodiment, the crystallization buffer in step (a) of the above process is selected from the group consisting of Tris-HCl, HEPES, acetate, phosphate, citrate, borate, imidazole and glycine. Alternatively, the crystallization buffer is selected from the group consisting of Tris-HCl, glycine, HEPES, imidazole, bis-Tris, AMP (2-amino-2 methylpropanol), AMPD (2-amino-2-methyl-1, 3-propanediol), AMPSO (3- ([1, 1-dimethyl-2-hydroxyethyl ] amino) -2-hydroxypropanesulfonic acid), bicine, ethanolamine, glycylglycine, TAPS, taurine (Taurin)

A Triane, and mixtures thereof. In another preferred embodiment, the crystallization buffer in the solution of step (a) is present at a concentration of between about 10mM and about 800 mM.

In another embodiment of the above method, the crystallization buffer in step (a) is present at a pH between about 3 and about 10. In a preferred embodiment, the crystallization buffer is present at a pH between about 6 and about 9. In yet another preferred embodiment, the crystallization buffer is present at a pH between about 7.5 and about 10.

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

In preferred embodiments of those methods comprising any step after step (b) of adding a precipitating agent to the crystallization solution, the precipitating agent is a non-ionic small molecule or a non-ionic polymer. In a preferred embodiment, the non-ionic polymer is selected from the group consisting of polyethylene glycol (PEG), polyvinyl alcohol, and mixtures thereof. In another preferred embodiment, the PEG has a molecular weight selected from the group consisting of between about 200 and about 8000, about 6000, about 4000, and about 3350. In yet another preferred embodiment, the PEG is present in the crystallization solution at a concentration between about 0.5% and about 20% (w/v). In another preferred embodiment of the above method, the precipitating agent is selected from the group consisting of amino acids, peptides, polyamino acids and mixtures thereof.

In another preferred embodiment, in step (c) of the above method, the ionic polymer is selected from the group consisting of protamine, polyarginine, polylysine, polyornithine and octaarginine (octarginine). In another preferred embodiment, in step (c) of the above method, polyarginine is added in a ratio of hGH to polyarginine of between 5: 1 and about 1: 25. The resulting crystallization solution is incubated at a temperature between about 15 ℃ and about 37 ℃ for about 16 to about 48 hours.

The present invention also provides yet another method of preparing crystals of human growth hormone, crystals of a human growth hormone derivative or a composition comprising such crystals and an excipient. The method comprises the following steps: (a) mixing a solution of human growth hormone or a human growth hormone derivative with a crystallization buffer to produce a crystallization solution; (b) adding deionized water to the crystallization solution; (c) adding a precipitating agent to the crystallization solution; (d) adding a salt to the crystallization solution; (e) incubating the crystallization solution at a temperature between about 10 ℃ and about 40 ℃ for about 2 to about 168 hours until crystals of human growth hormone or a human growth hormone derivative are formed, and (f) adding an ionic polymer to the crystals of human growth hormone or a human growth hormone derivative. According to one embodiment of the above process, step (f) is optional. In a preferred embodiment, the crystallization solution of step (e) is incubated at a temperature between about 15 ℃ and about 37 ℃ for about one to about two days. In another preferred embodiment, the crystallization solution of step (e) is incubated at a temperature between about 4 ℃ and about 37 ℃ for about one to about two days. In a further embodiment of the invention, the incubation is carried out for about 2 to about 48 hours. In another preferred embodiment, the crystallization solution of method step (e) above is incubated at a temperature between about 4 ℃ and about 40 ℃ for about 4 to about 48 hours until crystals of hGH or hGH derivative are formed.

In a preferred embodiment according to the present invention, in step (d) of the above process, the salt is a calcium salt or a monovalent cation salt. In a more preferred embodiment, the calcium salt is selected from the group consisting of calcium acetate, calcium chloride, calcium gluconate and calcium sulfate. In yet a more preferred embodiment, the calcium salt is calcium acetate. In a preferred embodiment, the calcium acetate is in the form of an aqueous solution having a pH between about 3 and about 9.0. In a more preferred embodiment, the aqueous calcium acetate solution has a pH between about 7.0 and about 8.6. In another embodiment, the calcium acetate in the crystallization solution of step (e) is present at a concentration between about 0.1mM and about 205 mM. In a more preferred embodiment, the calcium acetate in the crystallization solution in step (e) is present at a concentration of between about 85mM and about 100 mM.

In a more preferred embodiment, the monovalent cation is selected from the group consisting of lithium, sodium, potassium and ammonium. In yet a more preferred embodiment, the monovalent cation is sodium. Similarly, in a more preferred embodiment, the monovalent cation salt is selected from the group consisting of sodium citrate, sodium phosphate and sodium acetate. In yet a more preferred embodiment, the monovalent cation salt is sodium acetate. In a preferred embodiment, the sodium acetate is in the form of an aqueous solution having a pH between about 3 and about 9.0. In a more preferred embodiment, the aqueous sodium acetate solution has a pH between about 7.0 and about 8.6. In another embodiment, the sodium acetate in the solution of step (e) is present at a concentration between about 0.5mM and about 800 mM. In a more preferred embodiment, the calcium acetate in the crystallization solution in step (e) is present in a concentration between about 100mM and about 500 mM. Alternatively, the concentration may also be between about 85mM and about 100 mM.

In yet another embodiment, the hGH or hGH derivative in the crystallization solution in step (e) of the above method is present in a concentration of between about 2mg/ml and about 17.5 mg/ml. In another preferred embodiment, the hGH or hGH derivative in the crystallization solution of step (e) is present in a concentration of between about 14.5mg/ml and about 15.5 mg/ml. In a further embodiment, the hGH or hGH derivative in the crystallization solution in step (e) of the above method is present in a concentration of between about 2mg/ml and about 100 mg/ml.

In a preferred embodiment, the crystallization buffer of step (a) of the above process is selected from the group consisting of Tris-HCl, HEPES, acetate, phosphate, citrate, borate, imidazole and glycine. In another preferred embodiment, the crystallization buffer in the solution of step (a) is present at a concentration of between about 10mM and about 800 mM.

In a preferred embodiment, in the above process step (c), the precipitating agent is a non-ionic small molecule or a non-ionic polymer. In a preferred embodiment, the non-ionic polymer is selected from the group consisting of polyethylene glycol (PEG), polyvinyl alcohol and mixtures thereof. In another preferred embodiment, the PEG has a molecular weight selected from the group consisting of between about 200 and about 8000, about 6000, about 4000 and about 3350. In yet another preferred embodiment, the PEG is present in the crystallization solution at a concentration of about 0.5% to about 20% (w/v). In another preferred embodiment, in the above method step (c), the precipitating agent is selected from the group consisting of amino acids, peptides, polyamino acids and mixtures thereof.

In another preferred embodiment, in the above method step (f), the ionic polymer is selected from the group consisting of protamine, polyarginine, polylysine, polyornithine and octaarginine. In another preferred embodiment, in step (f) of the above method, polyarginine is added in a ratio of hGH to polyarginine (mg: mg) of between about 5: 1 and about 1: 25. In an alternative embodiment, the hGH fraction is between about 1: 5 and about 1: 25: polyarginine (mg: mg) was added at a ratio of polyarginine. Incubating the resulting solution of step (f) at a temperature between about 15 ℃ and about 37 ℃ for about 16 to about 48 hours. The influence of the polymer on the crystal dissolution rate of hGH or hGH derivative is reflected by the number of washes required for complete dissolution. For complete dissolution, the control crystals required about 7 to about 13 washes, while for complete dissolution, the crystals prepared with poly-arginine required between about 30 to 90 identical washes of the dissolution buffer. Washing is the washing step in a continuous dissolution process (see example 5).

In an alternative embodiment of any of the above methods, the calcium salt or monovalent cation salt may be present in the crystallization solution at a concentration of between about 0.01M and about 1M or between about 25mM and about 205 mM. In an alternative embodiment of any of the above methods, the crystallization solution is incubated for a time and at a temperature selected from the group consisting of about 0.25 days to about two days at a temperature of about 33 ℃; about 0.25 days to about two days at a temperature of about 25 ℃ and about 0.25 days to about two days at a temperature of about 15 ℃.

The invention also includes a method of screening crystals of hGH or hGH derivatives for use in a therapeutic formulation. The steps of such methods include: (1) washing the crystals of hGH or hGH derivative with a dissolution buffer at a temperature of 37 ℃ (e.g., 2mg crystals and 1ml of dissolution buffer) and (2) determining the in vitro dissolution rate of the crystals of hGH or hGH derivative per wash in the dissolution buffer, wherein the in vitro dissolution rate of the crystals is between about 10 minutes and about 1500 minutes, between about 2% and about 16% of the crystals, each wash step in a continuous dissolution process is from about 2 minutes to about 32 minutes (see example 5). The in vitro dissolution rate of the crystals may also be between about 4% and about 10% of the crystals in about 15 minutes or between about 0.04 and about 0.32mg of the crystals in about 15 minutes.

For a better understanding of the present invention, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Brief description of the drawings

FIG. 1 illustrates hGH crystal growth in the presence of 860mM ammonium phosphate (pH8.9), as imaged by light microscopy. See example 1.

FIG. 2 illustrates hGH crystal growth in the presence of 390mM sodium citrate, as imaged by light microscopy. See example 2.

FIG. 3 illustrates hGH crystal growth in the presence of 600mM disodium phosphate and 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 3.

FIG. 4 illustrates hGH crystal growth and co-crystallization with protamine sulfate (1mg/ml) in the presence of 85mM calcium acetate and 100mM Tris-HCl (pH8.6) as imaged by light microscopy. See example 4.

Fig. 5 shows the solubility of hGH crystals generated from ammonium phosphate, sodium citrate, disodium phosphate and calcium acetate/protamine precipitant as a function of time and monitored at 280 nm. See example 5.

FIG. 6 illustrates hGH crystal growth in the presence of 10% (v/v) isopropanol, 85mM calcium acetate and 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 6.

FIG. 7 illustrates hGH crystal growth in the presence of 5% (v/v) isopropanol, 85mM calcium chloride and 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 7.

FIG. 8 illustrates hGH crystal growth in the presence of 10% (v/v) ethanol, 10% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 8.

FIG. 9 shows the solubility of hGH crystals formed according to examples 6-8, as monitored at 280nm as a function of time in divided units. See example 9.

FIG. 10 illustrates hGH crystal growth in the presence of 85mM calcium acetate, 2% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 10.

FIG. 11 illustrates hGH crystal growth in the presence of 500mM sodium acetate, 6% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 11.

FIG. 12 illustrates hGH crystal growth in the presence of 85mM calcium chloride, 6% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 12.

FIG. 13 illustrates hGH crystal growth and co-crystallization with protamine sulfate (1mg/ml) in the presence of 85mM calcium acetate, 6% (v/v) PEG-6000, 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 13.

FIG. 14 illustrates hGH crystal growth in the presence of 125mM calcium acetate, 6% (v/v) PEG-MME-6000 and 100mM Tris-HCl (pH8.6), as imaged by light microscopy. See example 14.

FIG. 15 shows the solubility of hGH crystals produced according to examples 10-14, as monitored at 280nm as a function of time in divided units. See example 15.

FIG. 16 shows serum levels (ng/ml) of commercial hGH (soluble hGH) and hGH (crystals of hGH) prepared according to example 10 in female Sprague-Dawley rats, sampled within 24 hours after a single subcutaneous administration of 2.5mg/kg dose of soluble or crystalline hGH per rat. Serum levels were measured at t-0, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours. See example 16.

FIG. 17 illustrates the dissolution profile of hGH crystals (formed in the presence of 85mM calcium acetate, 2% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6)) after addition of different amounts of protamine sulfate. These different formulations of hGH crystals were then added to the dissolution buffer and allowed to stand for 1 hour before the concentration (area) of soluble hGH in the supernatant was determined by RP-HPLC. See example 17.

FIG. 18A shows hGH crystal growth in the presence of 500mM sodium acetate, 6% v/v PEG-6000 and 100mM Tris-HCl (pH8.6), as imaged by TEM lengthwise. See example 18.

FIG. 18B illustrates hGH crystal growth in the presence of 500mM sodium acetate, 6% v/v PEG-6000 and 100mM Tris-HCl (pH8.6), as imaged by TEM cross section. See example 18.

FIG. 19A shows serum levels (ng/ml) of hGH in female Sprague-Dawley rats of groups 3-5 and 9, sampled within 168 hours (0-72 hours shown) after subcutaneous administration of 6.7mg/kg of hGH per rat per day for seven days or a single subcutaneous administration for seven days. See example 22 and tables 7-12.

Fig. 19B shows the weight gain of female Sprague-Dawley rats selected for formulations (groups 2, 4 and 9) within 8 days after subcutaneous administration of 6.7mg/kg dose of hGH per day (group 2) or a single subcutaneous administration of hGH on the first day of seven days (groups 4 and 9) per rat for seven days. See example 22 and table 13.

Fig. 20A shows the concentration of hGH in serum as a function of time for female young macaques, which were dosed daily subcutaneously with soluble hGH (group 1), sodium hGH crystals complexed with polyarginine (group 2) and sodium hGH crystals complexed with protamine (group 3) according to table 16. See example 23.

Fig. 20B shows the concentration of IGF-1 in serum as a function of time for female young macaques administered daily subcutaneously soluble hGH (group 1), sodium hGH crystals complexed with polyarginine (group 2) and sodium hGH crystals complexed with protamine (group 3) according to table 18. See example 23.

Fig. 21A shows the concentration of hGH in serum as a function of time for female young macaques administered daily subcutaneously soluble hGH (group 1), sodium hGH crystals complexed with protamine (3: 1 ratio hGH: protamine) (group 2) and sodium hGH crystals complexed with protamine (2: 1 ratio hGH: protamine) (group 3) according to table 20. See example 24.

FIG. 21B shows the concentration of IGF-1 in serum as a function of time for female young macaques administered daily subcutaneously soluble hGH (group 1), hGH sodium crystals complexed with protamine (hGH: protamine at a 3: 1 ratio) (group 2), and hGH sodium crystals complexed with protamine (hGH: protamine at a 2: 1 ratio) (group 3) according to Table 22. See example 24.

Fig. 22 illustrates the seven day growth of male Wistar rats, which were subcutaneously administered control (group 1, once daily for seven days), soluble hGH (groups 4 and 5, once daily for seven days) and crystalline hGH (groups 6, 7,9 and 10, once daily for seven days) according to table 25. See example 25.

Fig. 23 illustrates the weight gain (grams) induced daily for seven days in male Wistar rats subcutaneously administered control (groups 1, once daily for seven days), soluble hGH (groups 4 and 5, once daily for seven days) and crystalline hGH (groups 6, 7,9 and 10, once daily for seven days) in accordance with table 26. See example 25.

Detailed Description

The following materials were used in the examples set forth below.

Material

Commercially available recombinant human growth hormone (rhGH) is from bresgen Ltd. (Thebarton, Australia), polyethylene glycol with an average molecular weight of 4000 or 6000(PEG-4000 or PEG-6000) is from Hampton Research (Laguna Niguel, California) and protamine sulphate is available from Fisher from ICN Biomedicals Inc. Ammonium phosphate, Tris-HC1, sodium citrate, disodium phosphate, calcium acetate, calcium chloride, zinc acetate, HEPES, sodium chloride, potassium chloride, sodium azide, Isopropanol (IPA), ethanol, and polyethylene glycol monomethyl ether were each obtained from Fisher (Pittsburgh, Pa.). Sprague-Dawley rats were obtained from Charles River Laboratories (Worcester, MA) or from biological Research Laboratories, Inc. (Worcester, MA). Polyarginine was obtained from Sigma (st. louis).

Analytical techniques and assays

Reversed phase high performance liquid chromatography. Reversed phase high performance liquid chromatography (RP-HPLC) was obtained on an Agilent 1100 series HPLC (Palo Alto, CA) equipped with a C5, 5cm × 4.6mm, 3 μm column (Supelco, Bellefonte, Pa.). The samples were dissolved in lysis buffer (50mM HEPES pH7.2, 140mM NaCl, 10mM KC1 and 0.02% (v/v) NaN₃) Neutralized and filtered (0.2 μm) before injection. The elution profile was monitored at 214 and 280nm using a gradient method with solvents a and B. Solvent a consisted of 99.9% deionized water/0.1% TFA. Solvent B consisted of 99.9% acetonitrile/0.1% TFA. All chemicals were HPLC grade, obtained from Fisher. Elution was carried out using a gradient of 40-50% B for 0-2 min, 50-60% B for 2-12 min, and 12-1560-85% B for 15 min. A flow rate of 1ml/min and a column temperature of 35 ℃ were maintained by running. Data were analyzed using Agilent chemical workstation software (Palo Alto, CA).

The concentration of protamine or polyarginine in the sample formulation was determined using a gradient method of solvent a (99.9% deionized water/0.1% TFA) and B (99.9% acetonitrile/0.1% TFA). Elution was carried out using a gradient design of 95: 5 (A: B) for 0-2.5 min, 65: 35 (A: B) for 2.5-7.5 min, 25: 75 (A: B) for 7.5-15.5 min, 25: 75 (A: B) for 15.5-17.0 min, 95: 5 (A: B) for 17-17.1 min for 20 min at a flow rate of 1 ml/min. Elution of typical protamine or polyarginine was obtained at 6.2 minutes and intact hGH eluted at 14 minutes. AUC calculation was determined at 213 nm. The content (mg/ml) of protamine and/or polyarginine additives was calculated from a standard curve generated for each of the additives. This same method can be used to analyze excipients released from the complex.

The hGH related to degradation was determined by a separate but similar reverse phase method. For example, in C5Supelco DiThe analysis was performed on a scovery Bio Wide Pore column (5 cm. times.4.6 mm, 3 μm particle size, 30nm Pore size) maintaining a thermostat temperature of 37 ℃ throughout the run. Using a mixture with mobile phase A (20% ACN, 80% H)₂O, 0.1% TFA) and mobile phase B (20% ACN, 80% 2-propanol, 01% TFA) were monitored by gradient method for elution profile. The gradient system changed from 20% to 45% B in 0 to 5 minutes, from 45% to 55% B in 5 to 15 minutes, from 55% to 90% B in 15 to 15.1 minutes, 90% B was fixed up to 17 minutes and after this step, 20% B was reestablished up to 20 minutes.

Size exclusion chromatography. High performance size exclusion chromatography (SEC-HPLC) was obtained on an Agilent 1100 series HPLC (PaloAlto, CA) equipped with a TSK-Gel G2000SWXL column (part #; 08450, Tosoh Biosep LLC, Montgomeryville, Pa.) (7.8 mm. times.30 cm, 5 μm) and an Agilent 1100 series MWD (UV). The samples were dissolved in 0.2ml of lysis buffer and filtered with 0.2 μm before injection into an Agilent 1100 series temperature controlled autosampler. The elution profile was monitored at 214 and 280nm with a mobile phase of 50mM Tris-HC1, 150mM NaCl, 0.05% NaN₃pH 7.5. The solvent was degassed using an Agilent 1100 series degasser, maintaining the column temperature at 25 ℃.

UV-VIS absorption and optical microscopy. UV-VIS spectroscopy was obtained on a Beckman DU 740 spectrophotometer from Beckman Coulter inc, Fullerton, CA. Optical micrographs at magnifications of 40x to 400x were obtained by bright field imaging using an Olympus BX-51 microscope and with a Sony DXC-970MD3CCD color digital video camera using Image-Pro software from Media Cybernetics l.p., Silver Springs, Maryland.

Transmission Electron Microscopy (TEM). The TEM analysis was performed as follows. The hGH crystal suspension in the mother liquor was washed twice with water to remove excess mother liquor and then negatively stained with 0.5% uranyl acetate for 1 hour. The stained hGH crystal suspension (1-5. mu.L) was transferred onto copper TEM grids. Excess liquid runs off (wicked away) and the sample grid is briefly air dried. The TEM grid was transferred to the sample stage of a JEOL 1210 transmission electron microscope and images were collected using an 80KV electron beam. A very well organized lattice structure was observed within the crystal, with an orientation in line with the prism axis of the crystal.

Example 1

hGH was crystallized from ammonium phosphate. Commercially available hGH (50mg) was first dissolved in 15ml Tris-HC1(10mM, pH8.0) and dialyzed against 2X 4000ml Tris-HCl (10mM, pH8.0) using a Pierce dialyzer cartridge having a Molecular Weight Cut Off (MWCO) of 10,000. Protein concentration was adjusted by centrifugation at 4000rpm for 20-30 minutes using a Millipore concentrator (MWCO10,000). E.g. at 280nm/0.813(1mg/ml hGH A)₂₈₀0.813 absorption units) the concentration of hGH was found to be in the range of 30-45mg/ml, as measured by absorption. Deionized water was added to the solution to give a final protein concentration of 10-20 mg/ml. By adding ammonium phosphate (NH) to the solution₄H₂PO₄) (2.5M; pH8.9) so as to obtain 860mM NH₄H₂PO₄To form crystals of hGH. The solution was then incubated at 25 ℃ for 16 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The obtained crystals were found to have a length of about 8 to 15 μm and a crystallization yield of more than 90%. See fig. 1.

Example 2

hGH was crystallized from sodium citrate. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 17.5 mg/ml. Crystals of hGH were formed by adding sodium citrate (1.5M) to the solution so that a final concentration of 390mM sodium citrate was obtained. No pH adjustment is required, except for hGH, which is already in 10mM Tris-HCl. The solution was then incubated at 25 ℃ for 16 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The obtained crystals were found to be less than 8 μm in length and to have a crystallization yield of more than 85%. See fig. 2.

Example 3

hGH was crystallized from sodium phosphate. Purification and concentration as described in example 1 was commercially availableThe obtained hGH. To the concentrated hGH solution was added deionized water to give a final protein concentration of 12.5-17.5 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. By adding disodium hydrogen phosphate (Na) to the solution₂HPO₄) (1M) formation of crystals of hGH so that 600mM Na was obtained₂HPO₄To the final concentration of (c). The solution was then incubated at 25 ℃ for 16 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The obtained crystals were found to be between 5 and 25 μm in length and the crystallization yield was more than 75%. See fig. 3.

Example 4

hGH was crystallized from calcium acetate and protamine sulfate. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 15 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. To the solution protamine sulphate was added to a final concentration of 1 mg/ml. Crystals of hGH were formed by adding calcium acetate (1M) to the solution so that a final concentration of 85mM calcium acetate was obtained. The solution was then incubated at 37 ℃ for 8 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The obtained crystals were found to be less than 20 μm in length and to have a crystallization yield of more than 70%. See fig. 4.

Example 5

Solubility characteristics of hGH crystals prepared by salt induced crystallization. After incubating the crystallization solutions of examples 1-4, crystals were precipitated and the remaining supernatant was removed. The crystal pellet (0.4mg) was resuspended in 0.200ml of lysis buffer (50mM HEPES (pH7.2), 140mM NaCl, 10mM KC1 and 0.02% (v/v) NaN) by pipetting or vortexing₃) Then equilibrated at 37 ℃ for about 15 minutes. The sample was then centrifuged at 10,000Xg for about 2 minutes and the supernatant removed completely for protein concentration determination by RP-HPLC, SEC-HPLC or UV-VIS measurement at 280 nm. The crystal pellet was further resuspended in 0.200ml of lysis buffer and the above procedure was repeated until no detectable protein was measured in the supernatant. This process is called continuous dissolution.

FIG. 5 shows the use of a monovalent (Na or NH) source in examples 1-4 above₄) Or the solubility behavior as a function of time in minutes of different hGH crystals prepared from divalent (Ca) salts. hGH dissolution was plotted as cumulative percent release derived from RP-HPLC, where AUC values of protein samples were measured in mg/ml using a UV-VIS spectrophotometer. The data show that hGH crystals prepared by addition of 390mM sodium citrate were completely dissolved after 60 minutes. In addition, by adding 600mM Na₂HPO₄Or 860mM NH₄H₂PO₄The prepared hGH crystals were completely dissolved after 60 or 75 minutes, respectively. On the other hand, hGH crystals prepared by adding 85mM calcium acetate and protamine sulfate were completely dissolved after 390 minutes (see Table 1 below).

TABLE 1 continuous dissolution test of hGH salts in dissolution buffer measured at 280nm Expressing the protein concentration as a percentage of the total released

Time (minutes)	390mM sodium citrate (example 2)	600mMNa₂HPO₄(example 3)	860mMNH₄H₂PO₄(example 1)	85mM calcium acetate + protamine (example 4)
Time (minutes)	390mM sodium citrate (example 2)	600mMNa₂HPO₄(example 3)	860mMNH₄H₂PO₄(example 1)	85mM calcium acetate + protamine (example 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	90	100.00	100.00	100.00	42.22
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

Example 6

hGH was crystallized from calcium acetate and 10% isopropanol. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 15 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. Crystals of hGH were formed by adding calcium acetate (1M) to the solution so that a final concentration of 85mM calcium acetate was obtained. To this solution, 10% (v/v) isopropyl alcohol (IPA) was added. The solution was then incubated at 25 ℃ for 16 hours. Rod-shaped crystals were obtained and imaged by optical microscopy. The crystal length obtained was found to be greater than 100 μm with a crystallization yield greater than 85%. See fig. 6.

Example 7

Crystallization from calcium chloride and 5% isopropanolhGH. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 15 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. By adding calcium chloride (CaCl) to the solution₂) (1M) so that a final concentration of 85mM calcium chloride was obtained, giving crystals of hGH. To this solution, 5% (v/v) isopropyl alcohol (IPA) was added. The solution was then incubated at 25 ℃ for 16 hours. Rod-shaped crystals were obtained and imaged by optical microscopy. The crystal length obtained was found to be greater than 200 μm with a crystallization yield greater than 85%. See fig. 7.

Example 8

hGH was crystallized from 10% PEG-6000 and 10% ethanol. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 25 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. Crystals of hGH were formed by adding 10% (v/v) PEG-6000 and 10% (v/v) ethanol (EtOH) to the solution. The solution was then incubated at 37 ℃ for 16 hours. Rod-shaped crystals were obtained and imaged by optical microscopy. The obtained crystals were found to be less than 25 μm in length and the crystallization yield was more than 70%. See fig. 8.

Example 9

Solubility characteristics of hGH crystals prepared with alcohol. After incubating the crystallization solutions prepared in examples 6 to 8, crystals were precipitated and the remaining supernatant was removed. The crystal pellet was resuspended in 0.200ml of lysis buffer (see example 5) by pipetting or vortexing and then equilibrated at 37 ℃ for about 15 minutes. The sample was then centrifuged at 10,000Xg for 2 minutes and the supernatant removed for protein concentration determination by RP-HPLC, SEC-HPLC or UV-VIS measurement at 280 nm. hGH dissolution was measured as cumulative percentage and derived from AUC values or UV-VIS mg/ml measurements. The crystal pellet was further resuspended in lysis buffer and the above procedure was repeated until no detectable protein was measured in the supernatant.

FIG. 9 and Table 2 illustrate the solubility behavior of hGH crystals prepared with 10% IPA/85mM calcium acetate, 5% IPA/85mM calcium chloride and 10% ethanol/10% PEG-6000 as a function of time in divided units. The results confirmed that hGH crystals prepared by adding 390mM sodium citrate were completely dissolved after 60 minutes. In addition, hGH crystals prepared by adding 10% IPA/85mM calcium acetate were completely dissolved after 150 minutes, while hGH crystals prepared by adding 5% IPA/85mM calcium chloride and 10% ethanol/10% PEG-6000 were completely dissolved after 120 minutes and 135 minutes, respectively.

TABLE 2 in vitro dissolution results of hGH crystals prepared with ethanol (examples 6-8) Protein concentration was measured at 280nm and expressed as a percentage of total released

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

Example 10

hGH was crystallized from calcium acetate and 2% PEG-6000. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 15 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. To this solution was added 2% (v/v) PEG-6000. Crystals of hGH were formed by adding calcium acetate (1M) to the solution so that a final concentration of 85mM calcium acetate was obtained. The solution was then incubated at 25 ℃ for 16 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The crystal lengths obtained were found to be between about 25 and about 75 μm with a crystallization yield of greater than 85%. See fig. 10.

Example 11

hGH was crystallized from sodium acetate and 6% PEG-6000. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 15 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. To this solution, 6% (v/v) PEG-6000 was added. Crystals of hGH were formed by adding sodium acetate (1M) to the solution so that a final concentration of 500mM sodium acetate was obtained. The solution was then incubated at 25 ℃ for 16 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The crystal lengths obtained were found to be between about 25 and about 75 μm with a crystallization yield of greater than 85%. See fig. 11.

Example 12

hGH was crystallized from calcium chloride and 6% PEG-6000. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 15 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. To this solution, 6% (v/v) PEG-6000 was added. By adding CaCl to the solution₂(1M) so as to obtain 85mM CaCl₂To form crystals of hGH. The solution was then incubated at 25 ℃ for 16 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The obtained crystals were found to have a length of more than 100 μm and a crystallization yield of more than 90%. See fig. 12.

Example 13

hGH was crystallized from calcium acetate, 6% PEG-6000 and protamine sulfate. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 15 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. To this solution, protamine sulfate (1mg/ml) and 6% PEG-6000(v/v) were added. Crystals of hGH were formed by adding calcium acetate (1M) to the solution so that a final concentration of 85mM calcium acetate was obtained. The solution was then incubated at 37 ℃ for 16 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The obtained crystals were found to be less than 25 μm in length and the crystallization yield was more than 70%. See fig. 13.

Example 14

hGH was crystallized from calcium acetate and 6% PEG-MME-5000. Commercially available hGH was purified and concentrated as described in example 1. To the concentrated hGH solution was added deionized water to give a final protein concentration of 15 mg/ml. Tris-HCl (1M, pH8.6) was added to a final concentration of 100 mM. To this solution, polyethylene glycol monomethyl ether (PEG-MME-500) was added. Crystals of hGH were formed by adding calcium acetate (1M) to the solution so that a final concentration of 125mM calcium acetate was obtained. The solution was then incubated at 25 ℃ for 16 hours. Needle-shaped crystals were obtained and imaged by optical microscopy. The obtained crystals were found to be less than 50 μm in length and to have a crystallization yield of more than 90%. See fig. 14.

Example 15

Solubility characteristics of hGH crystals prepared with polyethylene glycol. After incubating the crystallization solutions prepared in examples 10 to 14, crystals were precipitated and the remaining supernatant was removed. The crystal pellet was resuspended in 0.2ml of lysis buffer (see example 5) by pipetting or vortexing and then equilibrated at 37 ℃ for about 15 minutes. The sample was then centrifuged at 10,000Xg for 2 minutes and the supernatant removed for protein concentration determination by RP-HPLC, SEC-HPLC or UV-VIS measurement at 280 nm. The crystal pellet was further resuspended in lysis buffer and the above procedure was repeated until no detectable protein was measured in the supernatant.

FIG. 15 and Table 3 illustrate the use of 2% PEG-6000/85mM calcium acetate, 6% PEG-6000/500mM sodium acetate, 6% PEG-6000/85mM CaCl₂hGH crystals prepared with 6% PEG-6000A/85mM calcium acetate/protamine and 6% PEG-MME-5000/125mM calcium acetate as solubility behavior as a function of time divided into units. hGH dissolution was measured as cumulative percentage and derived from AUC values or UV-VIS mg/ml measurements. The results confirmed that hGH crystals prepared by adding 6% PEG-6000/85mM calcium acetate/protamine were the slowest to dissolve, and complete dissolution occurred after 495 minutes. The other crystals with the addition of 2% PEG-6000/85mM calcium acetate crystals dissolved at 300 minutes or the other hGH crystals dissolved in less time.

TABLE 3 PEG and hGH salts measured at 280nm in solutionContinuous dissolution test in buffer Protein solubility expressed as a percentage of the total amount released

Time (minutes)	2% PEG-6000/85mM calcium acetate (example 10)	6% PEG-6000/500mM sodium acetate (example 11)	6% PEG-6000/85mM calcium chloride (example 12)	6% PEG-6000/85mM calcium acetate/protamine sulfate (example 13)	6% PEG-MME-5000/125mM calcium acetate (example 14)
Time (minutes)	2% PEG-6000/85mM calcium acetate (example 10)	6% PEG-6000/500mM sodium acetate (example 11)	6% PEG-6000/85mM calcium chloride (example 12)		6% PEG-MME-5000/125mM calcium acetate (example 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

Example 16

Pharmacokinetic studies using Sprague-Dawley rats. 24 female Sprague-Dawley rats were dosed subcutaneously with 2.5mg/kg of soluble (commercially available) or crystalline (85mM calcium acetate/2% PEG-6000) hGH (prepared as described in example 10) suspensions. The average body weight of each rat was 200 g. The 24 rats were divided into two groups. Each group included subsystems of 3 groups of 4 rats each. Bleeding was collected via the jugular vein catheter implant at three specific time points in each subsystem. Due to the limited amount of blood that can be drawn at a given point in time, a leap frog design is used. To maintain the stability of the animals, the animal subsystems within the group bleed at the annotated time points. The serum samples were then edited to form a time line of linear progression (timeline). Standard deviation was determined by variation of serum levels in the subsystem from the mean of the subsystem at a given time point. See tables 4-6. In tables 4-5, designated animals 1-12 received soluble hGH and animals 13-24 received crystallized hGH, at a dose of 500. mu.g per animal.

FIG. 16 illustrates the levels of soluble and crystalline hGH in serum as a function of time. The half-life of crystalline hGH is almost 19 times higher than that of soluble hGH. For crystallized hGH, the time for maximum hGH to appear in serum was 4 hours, while for crystallized hGHDissolved hGH was 0.5 hours. Assuming groups and sub-systems of rats treated with soluble hGH or crystallized hGH at a concentration of 5.5mg/ml, equal to a dose of 2.2mg/kg, the Cmax values listed below in table 6 show that hGH delivered in crystalline form significantly reduces the maximum serum concentration compared to the same soluble dose. In addition, AUC of soluble hGH on total serum levels of hGH crystals was similar, indicating that crystallization did not significantly affect bioavailability. T for calculation of soluble and crystalline results_90％The value is obtained. This parameter indicates the time at which 90% of the total AUC occurred. Higher T_90％Values indicate that the drug remains in the serum for a longer period of time. T contained in Table 6_90％The results clearly show that the crystalline form leads to elevated hGH levels for a significantly longer time than the soluble form.

TABLE 4 results of the pharmacokinetics studies of soluble hGH in animals

Animal(s) production	Time to exsanguination (hours)	Average hGH (ng/ml) in serum	Standard deviation of the mean
Animal(s) production	Time to exsanguination (hours)	Average hGH (ng/ml) in serum	Standard deviation of the mean	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
	Total ═	3047.50		9-12	24	0.00	0.00

TABLE 5 preparation of crystalline hGH (85mM calcium acetate/2% PEG-6000) Results of the pharmacokinetic study of hGH animals

Animal(s) production	Time (hours)	Average hGH (ng/ml) in serum	Standard deviation of the mean
Animal(s) production	Time (hours)	Average hGH (ng/ml) in serum	Standard deviation of the mean	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
	Total ═	1386.379		21-24	24	2.69	0.74

TABLE 6 pharmacokinetic parameters based on the data in TABLE 4 and TABLE 5

	Soluble in	Of crystallisation
	Soluble in	Of crystallisation	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 (hours)	0.5	9.4
C_max(ng/ml)	1172	334	Half-life (hours)	0.5	9.4
C_max(ng/ml)	1172	334	T_max(hours)	0.5	4
AUC(o-t)(ng/hr/ml)	2819	2472	T_max(hours)	0.5	4
AUC(o-t)(ng/hr/ml)	2819	2472	AUC(2)	2819	2508
T_90％(hours)	4	10	AUC(2)	2819	2508

Example 17

Effect of protamine sulfate on the dissolution profile of hGH crystals. FIG. 17 illustrates the dissolved amount of hGH crystals prepared according to example 10(85mM calcium acetate, 2% (v/v) PEG-6000 and 100mM Tris-HCl (pH8.6)) after adding a given amount of protamine sulfate to a pre-existing solution of hGH calcium crystals after incubation for 1 hour in a dissolution buffer at 37 ℃. The ratio of HGH to protamine (mg: mg) is shown in FIG. 17. The figure illustrates that protamine significantly affects the dissolution of hGH crystals.

Example 18

hGH was crystallized from sodium acetate. Here, soluble recombinantly produced hgh (rhgh) frozen bulk feed solutions were obtained from two stocks-one from e.coli (Novartis) and the other from yeast (Lucky Gold). Independent analysis of rhGH from e.coli and yeast stock solutions, regardless of its source, yielded rhGH with identical crystallization and solubility characteristics. Approximately 3.3ml (supplied with 10-20mg/ml rhGH in unknown buffer) of thawed rhGH feed solution was purified using a 10 DG-desalting column supplied by BioRad. Before sample loading, the column was treated by washing the column with 30ml Tris-HCl (10mM, pH 8.0). The rhGH sample was then loaded and allowed to enter the column by gravity. After discarding the first 3ml eluate, another 5ml 10mM Tris-HCl pH8.0 was added. 4.5ml of desalted rhGH was eluted and collected. Concentration was then performed by centrifugation using a Millipore concentrator (MWCO10,000) at 3500rpm for 20-30 minutes. The concentration of hGH was in the range of 30mg/ml as determined by absorbance at 280nm/0.813(1mg/ml hGHA280 ═ 0.813 absorbance units). Crystals were produced by adding deionized water, Tris-HCl (pH8.6), PEG-6000 and sodium acetate to a final concentration of 100mM, 6% (v/v) and 500mM, respectively, protein 15mg/ml in the total solution. The solution was then gently mixed and incubated at 33 ℃ for 12-16 hours. Needle or rod shaped crystals were obtained and TEM imaged (fig. 18A and 18B). The crystal length varies from about 2 to 25 μm. After centrifugation and pellet precipitation of the crystals, the supernatant was extracted and the crystallization yield was measured to be greater than 85%. Crystals can also form at temperatures between 33 ℃ and 15 ℃, but require increased crystallization time and may result in a reduction in yield.

Example 19

Complexation of sodium hGH with ionic polymer additives. After determination of the crystallization yield (see example 18), rhGH sodium crystals were resuspended in mother liquor (250mM NaOAc, 25mM Tris-HCl (pH8.6), 6% PEG-6000, and 7mg/ml protamine sulfate or 4.2mg/ml polyarginine) so as to reach a final concentration of 21mg/ml rhGH sodium crystals. The protein to additive ratio was about 3: 1 (mg: mg) for rhGH to protamine sulfate and 5: 1 (mg: mg) for rhGH to polyarginine sulfate. These ratios were calculated as molar ratios of about 1: 1.715 for rhGH to protamine and about 1: 0.587 for rhGH to polyarginine. The rhGH pellets were resuspended homogeneously in the appropriate mother liquor and incubated overnight at 2-8 deg.C before centrifugation to obtain condensed pellets. The supernatant was removed and the pellets were resuspended in the same mother liquor (without ionic polymer additive) and stored at 4 ℃.

Additional rhGH to polymer additive ratios were obtained by varying the additive concentration (mg/ml) of the mother liquor but still resuspending to 21mg/ml rhGH. For example, an increased concentration of protamine sulfate in the mother liquor (10.5mg/ml) can be used to obtain a 2: 1 rhGH: additive ratio after resuspension.

Example 20

rhGH was crystallized from zinc acetate. rhGH was crystallized from zinc acetate and acetone. Approximately 3.3ml (10-20mg/ml) of thawed rhGH feed solution was purified using a 10 DG-desalting column supplied by BioRad. Before sample loading, by using 30ml Na₂HPO₄/NaH₂PO₄The column was treated by washing the column (10mM, pH 6.1). The rhGH sample was then loaded and allowed to enter the column by gravity. After discarding the first 3ml eluate, another 5.0ml 10mM Na was added₂HPO₄/NaH₂PO₄pH 6.1. A4.5 ml aliquot of desalted rhGH was eluted and collected. Concentration was then performed by centrifugation at 3500rpm for 5-10 minutes using a Millipore concentrator (MWCO10,000). The concentration of hGH was in the range of 15mg/ml as determined by absorbance at 280nm/0.813(1mg/ml hGH a280 ═ 0.813 absorbance units). By adding 400. mu.l of deionized water, 8.91mM Na₂HPO₄/NaH₂PO₄pH6.1, 0.88mg/ml Zinc acetate, 9.89% acetone to 100. mu.l in 10mM Na₂HPO₄/NaH₂PO₄(pH6.1) to produce crystals. The solution was then gently mixed and incubated at 15 ℃ for 24-48 hours. Hexagonal-like crystals were obtained, varying in width from about 2 to 25 μm. After centrifugation and pellet formation of the crystals, the supernatant was extracted and the crystallization yield was measured to be approximately 55%.

Example 21

Crystallization of hGH using calcium acetate and complexation of the calcium hGH with the ionic polymer additive (polyarginine). Here, frozen bulk feed solutions of soluble recombinantly produced hGH (rhGH) were obtained from two stocks-one from E.coli (Novartis) and the other from yeast (Lucky Gold). Approximately 3.5ml (12 mg/ml rhGH in Tris-HCl (10mM, pH 8.0)) of thawed rhGH feed solution was purified using a 10 DG-desalting column supplied by BioRad. Before sample loading, the column was treated by washing the column with 30ml Tris-HCl (10mM, pH 8.0). The rhGH sample was then loaded and allowed to enter the column by gravity. After discarding the first 3ml eluate, another 5.0ml 10mM Tris-HClpH8.0 was added. 4.5ml of desalted rhGH was eluted and collected. Concentration was then performed by centrifugation at 3500rpm for 20-30 minutes using a Millipore concentrator (MWCO10,000). The concentration of hGH was in the range of 30mg/ml as measured by absorbance at 280nm/0.813(1mg/ml hGH a280 ═ 0.813 absorbance units). Crystals were produced by adding 1M Tris-HCl (pH8.6), 50% PEG-6000 and 1M calcium acetate to a rhGH 30mg/ml stock solution so as to obtain final concentrations of 15mg/ml rhGH, 100mM Tris-HCl (pH8.6), 2% (v/v) PEG-6000 and 85mM calcium acetate. The solution was then gently mixed and incubated at 33 ℃ for 12-16 hours. Needle-shaped crystals with a length ranging from about 2 to 25 μm were obtained. After extracting the supernatant and centrifuging and pelleting the crystals, the crystallization yield was measured to be greater than 85%. The crystals can also form at temperatures between 33 ℃ and 15 ℃, but require increased crystallization time and reduced yield. After determination of the crystallization yield (see example 18), rhGH calcium was resuspended in formulation media (5mM CaOAc, 100mM Tris-HCl (pH8.6), 6% PEG-6000 and 4.2mg/ml polyarginine) so as to reach a final concentration of 21mg/ml rhGH calcium. For rhGH and polyarginine, the ratio of protein to additive was 5: 1 (mg: mg). These ratios are calculated to be about a 10: 0.587 molar ratio for rhGH to polyarginine. The rhGH pellets were resuspended homogeneously in a suitable mother liquor and incubated overnight at 2-8 ℃ before centrifugation to obtain condensed pellets. The supernatant was removed and the pellets were resuspended in the same mother liquor without ionic additive and stored at 4 ℃.

Example 22

Pharmacokinetic and pharmacodynamic studies of hGH and hGH dication crystals administered subcutaneously using Sprague-Dawley rats. The aim of this study was to evaluate the controlled release of hGH from hGH crystal suspensions and the increased body weight following subcutaneous implantation of hGH crystal suspensions in pituitysectomized Sprague-Dawley rats. The study design is as follows:

TABLE 7 study design

Group # or test Compounds	Sample (I)^a	Description of the samples	# of rat	Dose level (mg/rat/week)	Dosing regimens
Group # or test Compounds	Sample (I)^a	Description of the samples	# of rat	Dose level (mg/rat/week)	Dosing regimens	1	Soluble media	Buffer in Water for injection (WFI)^b	3	--	100 μ l once a day for 7 days
2	Is soluble every day	Commercially available hGH in WFI^c(1.5mg/ml)	9	1.05	100 μ l once a day for 7 days	1	Soluble media	Buffer in Water for injection (WFI)^b	3	--	100 μ l once a day for 7 days
2	Is soluble every day	Commercially available hGH in WFI^c(1.5mg/ml)	9	1.05	100 μ l once a day for 7 days	3	Calcium acetate, PEG, protamine	See example 10- (21mg/ml, diluted before injection and combined with mother liquor 1^dMild mix 1: 1)	9	1.05	100 μ l once the first day
4	Calcium acetate, PEG	See example 13- (21mg/ml, diluted before injection and combined with mother liquor 1^dMild mix 1: 1, hGH: protamine ratio 5: 1)	9	1.05	100 μ l once the first day	3	Calcium acetate, PEG, protamine		9	1.05	100 μ l once the first day
4	Calcium acetate, PEG		9	1.05	100 μ l once the first day	5	Zinc acetate	See example 20- (21mg/ml, diluted before injection and combined with mother liquor 2^eMild mix 1: 1)	3	1.05	100 μ l once the first day
7	Medium	From groups 3, 4&5^fMother liquor of (2)	3	--	100 μ l once the first day	5	Zinc acetate		3	1.05	100 μ l once the first day
7	Medium	From groups 3, 4&5^fMother liquor of (2)	3	--	100 μ l once the first day	8	Medium	Mother liquor from group 9	3	--	100 μ l once the first day
9	Calcium acetate, PEG, polyarginine	See example 21- (21mg/ml, diluted before injection and combined with mother liquor 1^dMild mix 1: 1, hGH: Polyarginine ratio 12: 1)	9	1.05	100 μ l once the first day	8	Medium	Mother liquor from group 9	3	--	100 μ l once the first day

^aAll samples were stored at 4 ℃ and, if desired, warmed to room temperature for 30 minutes before dilution with the formulation medium defined in d, and injected into the indicated number of rats.

^bThe buffer contained 7.5mg/ml D-mannitol, 12mg/ml sucrose.

^cCommercially available hGH is available from BesaGen ltd.

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

^eZinc acetate, NaH₂PO₄(pH6.1) and ethanol.

^fA single formulation vehicle as defined in d was used as a vehicle control for all these groups.

Upon arrival, 80 female Sprague-Dawley rats, weighing approximately 150 g + -25 g and approximately 4-6 weeks of age, were individually housed under controlled conditions (approximately 21 + -3 deg.C temperature, 50 + -20% relative humidity, 12 hours of light and 12 hours of darkness in each 24 hour period, 10-15 air exchanges per hour) and allowed free access to pure water and laboratory food throughout the study. Rats were allowed to acclimate for one week prior to testing.

Of 80 rats, 48 were administered hGH suspensions according to Table 7. Test compounds were administered subcutaneously in a single bolus over the dorsal site once the first day or once daily for seven consecutive days. Injection sites were shaved and marked up to three days prior to injection, after which time as needed to facilitate injection. Test compounds were administered using a 30-gauge x 8mm needle attached to a 300 μ l syringe. Before being drawn into the syringe and again before administration, the test compound was carefully inverted to ensure suspension or solution homogeneity without causing foaming. The injection volume was approximately 0.1ml per rat.

Blood samples were collected from rats in groups 1,5, 7 and 8 (each group had 3 rats) at hours 4, 32, 96 and 168 after the first day injection. Blood samples from groups 2, 3, 4 and 9 (each group having 9 rats) rats were further subdivided into 3 groups of 3 rats. Here, blood samples were taken from a first subset of rats at 0.5, 24, 72 and 168 hours after the first day injection, from a second subset of rats at 4, 32, 96 and 168 hours, and from a third subset of rats at 8, 48, 120 and 168 hours. Usually through the orbital sinus without anesthesia or CO₂/O₂Bleeds were collected on anesthetized rats and collected in BD Microtainer tubes with serum separating agent. The samples were then centrifuged at about 4 ℃ and the serum recovered and stored frozen (about-80 ℃) prior to determination of hGH and IGF-1 levels.

Serum samples were then pooled and in the case of groups 2, 3, 4 and 9, a linear progressive timeline (timeline) was formed. Standard deviation was determined by variation of serum levels within a subset at a given time point from the mean of the subset. See tables 8-14 and FIG. 19A. Serum levels of rhGH (ng/ml) were shown when administered in the particular crystal formulation. Animals were bled at specific time points corresponding to dosing according to the study protocol. The results clearly show that there is a difference between the absorption of complexed crystalline material (e.g. protamine and polyarginine) and the uncomplexed crystalline formulation (e.g. CaOAC and ZnOAC).

Table 8 results of the hGH animal pharmacokinetic study, group 2: is soluble every day

Animal(s) production	Blood sampling time (hours)	Average hGH in serum (ng/ml)	Standard deviation of the mean
Animal(s) production	Blood sampling time (hours)	Average hGH in serum (ng/ml)	Standard deviation of the mean	All are	0	0	0
1-3	1	1262	18	All are	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 are	168	0	0
	Total ═	5174		All are	168	0	0

Table 9 results of the hGH animal pharmacokinetic study, group 3: calcium acetate, PEG

Animal(s) production	Blood sampling time (hours)	Average hGH in serum (ng/ml)	Standard deviation of the mean
Animal(s) production	Blood sampling time (hours)	Average hGH in serum (ng/ml)	Standard deviation of the mean	All are	0	0.00	0.00
1-3	0.5	1927	771	All are	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 are	168	0.00	0.00
	Total ═	8542		All are	168	0.00	0.00

TABLE 10 results of the pharmacokinetic studies of hGH animals, group 4: calcium acetate, PEG, protamine

Animal(s) production	Blood sampling time (hours)	Mean h in serumGH(ng/ml)	Standard deviation of the mean
Animal(s) production	Blood sampling time (hours)	Mean h in serumGH(ng/ml)	Standard deviation of the mean	All are	0	0.00	0.00
1-3	0.5	0.00	0.00	All are	0	0.00	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 are	168	0	0
	Total ═	2849		All are	168	0	0

Table 11 results of the hGH animal pharmacokinetic study, group 5: zinc acetate

Animal(s) production	Blood sampling time (hours)	Average hGH in serum (ng/ml)	Standard deviation of the mean
Animal(s) production	Blood sampling time (hours)	Average hGH in serum (ng/ml)	Standard deviation of the mean	All are	0	0.00	0.00
1	4	2417	767	All are	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 are	168	0	0
	Total ═	2418		All are	168	0	0

Table 12 results of the pharmacokinetic studies in hGH animals, group 9: calcium acetate, polyarginine

Animal(s) production	Blood sampling time (hours)	Average hGH in serum (ng/ml)	Standard deviation of the mean
Animal(s) production	Blood sampling time (hours)	Average hGH in serum (ng/ml)	Standard deviation of the mean	All are	0	0.00	0.00
1-3	1	502	75	All are	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 are	168	0.00	0.00
	Total ═	3980		All are	168	0.00	0.00

TABLE 13 pharmacokinetic parameters based on the data in tables 8-12

Group of	Every 7 days	C_max(ng/ml)	T_max(hours)
Group of	Every 7 days	C_max(ng/ml)	T_max(hours)	2: is soluble every day	6.7mg/kg	1262.70	1
3: calcium acetate, PEG	6.7mg/kg	5203.80	4	2: is soluble every day	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, polyarginine	6.7mg/kg	1036.63	8

Body weight of each rat was measured and recorded prior to injection on the first day of the study and prior to the time of blood draw on each successive morning of the study. Thus, the weight gain or loss of each rat in each group was calculated by subtracting the body weight on day 1 (before injection) from the body weight on each successive day (before injection). The average body weight of all rats in the daily group was calculated. These results are provided in table 14.

TABLE 14 daily weight gain or loss (g) in Sprague-Dawley rats

Group of	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8
Group of	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	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 and fig. 19B illustrate the seven day effect of administering a single dose (day 1) of the crystals of the present invention compared to the seven day effect of administering a daily dose of commercially available hGH. For example, fig. 19B demonstrates that hGH crystalline calcium complexed with polyarginine achieves a weight gain comparable to that of a soluble daily dose of only one dose over the same period of time. In comparing the respective release profiles of increased body weight and rhGH in serum, it is evident that a longer release of the poly-arginine formulation correlates with a more sustained rate of body weight gain.

Example 23

Comparative pharmacodynamic studies in female young macaques (jiufenile cynomologous monkeys). The objective of this study was to evaluate the in vivo pharmacokinetic profile of crystalline recombinant human growth hormone (rhGH) when administered subcutaneously to female young macaques. These data were generated to model the controlled release of crystalline rhGH in serum and the weight gain as a function of crystalline rhGH release.

TABLE 15 study design of primate study I

Group #	Sample (I)	Administration of doses (hours)	Dosage level (mg/kg)	Dosage concentration (mg/ml)	Dosage capacity (ml/kg)	Animal number (female)
Group #	Sample (I)	Administration of doses (hours)	Dosage level (mg/kg)	Dosage concentration (mg/ml)	Dosage capacity (ml/kg)	Animal number (female)	1	Is soluble every day^a	0，24，48，72，96，120，144	0.8	3.2	0.25	4
2	Sodium acetate, PEG, polyarginine^b	0	5.6	22.4	0.25	4	1	Is soluble every day^a	0，24，48，72，96，120，144	0.8	3.2	0.25	4
2	Sodium acetate, PEG, polyarginine^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) was obtained from Novarts and diafiltered in WFI. Group 1 (positive control) received soluble hGH on each dosing day.

^bSee examples 18 and 19 for preparation.

^cAll doses were delivered after daily blood draw.

12 female young macaques were divided into three groups of four animals and either soluble rhGH (group 1), rhGH sodium crystals with PEG and polyarginine (group 2, according to examples 18 and 19) or rhGH sodium crystals with PEG and protamine (group 3, according to examples 18 and 19) were administered. Monkeys weighing 2-6kg and aged 4-7 years at the start of treatment were individually housed in stainless steel cages equipped with automatic watering systems or water bottles. Animals were controlled in room environment (approximately 21 ± 3 ℃, 30-70% humidity, 12 hours light and 12 hours dark per 24 hour period, and 12-20 air exchanges per hour) and monkeys were fed twice daily with a standard Certified commercial Primate Diet (Harlan Teklad Certified Primate Diet # 2055C).

This primate study was performed in order to measure and compare the serum concentrations of hGH and IGF-1 after administration of soluble rhGH (group 1), rhGH sodium crystals with PEG and polyarginine (group 2) or rhGH sodium crystals with PEG and protamine (group 3). Body weights were recorded for all animals prior to transfer and dosing at the times indicated in table 15 above. Blood samples (approximately 1ml) were collected from each animal via femoral, brachial or saphenous vein on the morning of-216, -120, 0, 2, 4, 6, 8, 10, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288 and 312 days. Blood was collected in serum separation tubes, left at room temperature for 30-45 minutes to allow clotting, and centrifuged at 3000rpm2-8 ℃ for 10 minutes. Each serum sample was divided into 100. mu.l aliquots and the remaining aliquots, all of which were stored at-70. + -. 10 ℃ prior to analysis. Typically, the smaller the 100 μ l aliquot used in the rhGH assay, the larger the remainder used in the IGF-1 assay. There are some exceptions due to the replication volume required.

The collected serum samples were then analyzed for hGH concentration (see Table 16). rhGH concentrations falling outside the standard range were diluted appropriately. All values were used to obtain the mean background level for each individual animal of primate GH. The mean value for each animal was subtracted from the serum levels of the test subjects determined at each time point. The corrected values for each time point were then averaged to obtain a corrected average value of rhGH in serum. The standard error is then calculated by using the standard deviation of the corrected mean and divided by N to the square root of 4.

TABLE 16 group 1 (soluble daily), 2(rhGH sodium/polyarginine) And 3(rhGH sodium/protamine) rhGH levels

Time of day	Group 1-average daily soluble rhGH (ng/ml)	Standard error of	Group 2-average rhGH sodium/Polyarginine (ng/ml)	Standard error of	Group 3-average rhGH sodium/protamine (ng/ml)	Standard error of
Time of day	Group 1-average daily soluble rhGH (ng/ml)	Standard error of	Group 2-average rhGH sodium/Polyarginine (ng/ml)	Standard error of	Group 3-average rhGH sodium/protamine (ng/ml)	Standard error of	-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

Note: rhGH values are the average values from 4 animals, adjusted by baseline, i.e., values minus baseline. The baseline is the average of values at t-216, -120 and 0 hours.

Fig. 20A illustrates the level of rhGH in serum as a function of time in hours after baseline adjustment in groups 1, 2, and 3.

TABLE 17 summary of pharmacokinetic parameters based on the data in 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(hours)	2	10	10	C_max(ng/ml)	372	409	381
T_max(hours)	2	10	10	AUC(0-t)(ng.hr.kg/ml.mg)	4570	3503	3455
T_90％(hours)	20	74	77	AUC(0-t)(ng.hr.kg/ml.mg)	4570	3503	3455

^aCommercially available hGH (soluble, uncrystallized form) was diafiltered in WFI. Group 1 (positive control) received soluble hGH on each of 7 dosing days.

The above data confirm the time (T) for maximum hGH to appear in serum for the poly-arginine-complexed sodium hGH crystals_max) 10 hours for protamine complexed sodium hGH crystals and 2 hours for soluble hGH. Assuming delivery of soluble hGH in crystalline administration at 1/7th dose, C listed in Table 17 above_maxThe values show that when hGH is delivered in either of the two complexed crystalline forms, the initial serum concentration spike is significantly reduced. In addition, T for the soluble and crystalline groups was calculated_90％The value is obtained. T of group 1 (soluble form)_90％20 hours, and T of groups 2 and 3 (complexed crystalline form)_90％74 and 77 hours, respectively. These results clearly show that the complexed crystalline form results in elevated hGH levels being maintained for a significantly longer period of time than the soluble form.

In addition to determining the serum concentration of hGH, the level of IGF-1 was measured as a function of time. The efficacy of rhGH was determined by measuring the production of IGF-1. Table 18 below reports the IGF-1 concentrations for the animals in groups 1-3. Figure 20B illustrates that after subtraction of baseline endogenous IGF-1 levels, the complexed crystal formulation has demonstrated an ability to stimulate IGF-1 release comparable to daily soluble administration. These results in non-human primates show that the formulations of the invention can be advantageously used to achieve similar efficacy in humans.

TABLE 18 group (soluble daily rhGH sodium/polyarginine And rhGH sodium/protamine) are used in the treatment of cancer

Time of day	Group 1-average daily soluble IGF-l (ng/ml)	Standard error of	Group 2-average rhGH sodium/polyarginine IGF-1(ng/ml)	Standard error of	Group 3-average rhGH sodium/protamine IGF-l (ng/ml)	Standard error of
Time of day	Group 1-average daily soluble IGF-l (ng/ml)	Standard error of	Group 2-average rhGH sodium/polyarginine IGF-1(ng/ml)	Standard error of	Group 3-average rhGH sodium/protamine IGF-l (ng/ml)	Standard error of	-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

Note: IGF-1 values are reported as mean values calculated from 4 animals, adjusted by baseline, i.e., values minus baseline. The baseline is the average of values at t-216, -120 and 0 hours.

Example 24

Comparative pharmacodynamic studies in female young macaques with different protamine ratios. The objective of this study was to evaluate the in vivo pharmacokinetic profile of crystalline recombinant human growth hormone (rhGH) when administered subcutaneously to female young macaques. These data were generated to investigate the effect of the ratio of hGH sodium to protamine on the controlled release of crystalline rhGH in serum and the weight gain as a function of crystalline rhGH release.

TABLE 19 Experimental study of primate study II

Group #	Sample (I)	Administration of doses (hours)	Dosage level (mg/kg)	Dosage concentration (mg/ml)	Dosage volume (ml/kg)	Animal number (female)
Group #	Sample (I)	Administration of doses (hours)	Dosage level (mg/kg)	Dosage concentration (mg/ml)	Dosage volume (ml/kg)	Animal number (female)	1	Is soluble every day^a	0，24，48，72，96，120，144	0.8	3.2	0.25	4
2	Sodium acetate, PEG, protamine (3: l)^b	0	5.6	22.4	0.25	4	1	Is soluble every day^a	0，24，48，72，96，120，144	0.8	3.2	0.25	4
2	Sodium acetate, PEG, protamine (3: l)^b	0	5.6	22.4	0.25	4	3	Sodium acetate, PEG, protamine (2: l)^b	0	5.6	22.4	0.25	4

^bSee examples 18 and 19 for preparation.

^cAll doses were delivered after daily blood draw.

In primate study II, 12 female cynomolgus monkeys described in primate study I were divided into three groups, each group having four animals, and either soluble rhGH (group 1), rhGH sodium crystals with PEG and protamine (3: 1 rhGH: protamine) (group 2) (examples 18 and 19) or rhGH sodium crystals with PEG and protamine (2: 1 rhGH: protamine) (group 3) (examples 18 and 19) were administered. Monkeys weighing 2-6kg and aged 4-7 years at the start of treatment were individually housed in stainless steel cages equipped with automatic watering systems or water bottles. Animals were controlled in room environment (approximately 21 ± 3 ℃, 30-70% humidity, 12 hours light and 12 hours dark per 24 hour period, and 12-20 air exchanges per hour) and monkeys were fed twice daily with a standard certified commercial primate grain (Harlan Teklad certificated primate Diet # 2055C).

This primate study was performed in order to measure and compare the serum concentrations of hGH and IGF-1 after administration of soluble rhGH (group 1), rhGH sodium crystals with PEG and protamine (3: 1 rhGH: protamine) (group 2) and rhGH sodium crystals with PEG and protamine (2: 1 rhGH: protamine) (group 3). Body weights were recorded for all animals prior to transfer and dosing at the times indicated in table 19 above. Blood samples (approximately 1ml) were collected from each animal via the femoral, brachial or saphenous vein in the morning of-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. Blood was collected in serum separation tubes, left at room temperature for 30-45 minutes to allow clotting, and centrifuged at 3000rpm for 10 minutes at 2-8 ℃. Each serum sample was divided into 100. mu.l aliquots and the remaining aliquots, all of which were stored at-70. + -. 10 ℃ prior to testing.

hGH concentration (ng/ml) in the collected serum samples was analyzed and baseline corrected (see data in Table 20). Note that rhGH concentrations falling outside the standard range were diluted appropriately. All values were then used to obtain the mean background level for each individual animal of primate hGH. The mean value for each animal was subtracted from the serum levels of the test subjects determined at each time point. The corrected values for each time point were then averaged to obtain a corrected average value of rhGH in serum. The standard error is then calculated by using the standard deviation of the corrected mean and divided by N to the square root of 4.

TABLE 20 groups 1 (soluble daily), 2(rhGH sodium/protamine (3: 1)) And 3(rhGH sodium/protamine (2: 1)) rhGH levels

Time (hours)	Group 1-average daily soluble rhGH (ng/ml)	Standard error of	Group 2-average rhGH sodium/protamine (3: 1), (ng/ml)	Standard error of	Group 3-average rhGH sodium/protamine (2: 1) (ng/ml)	Standard error of
Time (hours)	Group 1-average daily soluble rhGH (ng/ml)	Standard error of	Group 2-average rhGH sodium/protamine (3: 1), (ng/ml)	Standard error of	Group 3-average rhGH sodium/protamine (2: 1) (ng/ml)	Standard error of	-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

Note: rhGH values are reported as mean values calculated from 4 animals, adjusted by baseline, i.e., values minus baseline. The baseline is the average of the values at t-144, -120, -96, -72, -48, -24, and 0 hours.

Fig. 21A illustrates the level of rhGH in serum as a function of time in hours after baseline adjustment in groups 1, 2, and 3.

TABLE 21 summary of pharmacokinetic parameters based on the data in 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(hours)	4	10	24	C_max(ng/ml)	401	456	380
T_max(hours)	4	10	24	AUC(0-t)(ng.hr.kg/ml.mg)	4432	3669	2893
T_90％To (hours)	20	119	72	AUC(0-t)(ng.hr.kg/ml.mg)	4432	3669	2893

These data confirm that the time for maximum hGH to appear in serum is 10 hours for protamine (3: 1) complexed hGH crystals, 24 hours for protamine (2: 1) complexed hGH crystals, and 4 hours for soluble hGH. Assuming delivery of soluble hGH in crystalline administration at 1/7th dose, C listed in Table 22 above_maxThe values show that the maximum serum concentration is significantly reduced when hGH is delivered in either of the two complexed crystalline forms. T of group 1 (soluble form)_90％20 hours, and T of groups 2 and 3 (complexed crystalline form)_90％119 and 72 hours respectively. These results clearly show that the complexed crystalline form results in elevated hGH levels being maintained for a significantly longer period of time than the soluble form.

In addition to determining the serum concentration of hGH, the level of IGF-1 was measured as a function of time. The efficacy of rhGH was determined by measuring the production of IGF-1. Table 22 below reports the IGF-1 concentrations for the animals in groups 1-3. Figure 21B illustrates that the complexed crystal formulation is able to stimulate IGF-1 release after subtraction of a baseline of endogenous IGF-1 levels, which is comparable to daily soluble administration. These non-human primate results show that the formulations of the present invention can be advantageously used to elicit similar efficacy in humans.

TABLE 22 group 1 (soluble daily), 2(rhGH sodium/protamine (3: 1)) And 3(rhGH sodium/protamine (2: 1)) IGF-1 levels

Time (hours)	Group 1-average daily soluble, IGF-1(ng/ml)	Standard error of	Group 2-average rhGH sodium/protamine (3: 1), IGF-1(ng/ml)	Standard error of	Group 3-average rhGH sodium/protamine (2: 1), IGF-1(ng/ml)	Standard error of
Time (hours)	Group 1-average daily soluble, IGF-1(ng/ml)	Standard error of	Group 2-average rhGH sodium/protamine (3: 1), IGF-1(ng/ml)	Standard error of	Group 3-average rhGH sodium/protamine (2: 1), IGF-1(ng/ml)	Standard error of	-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

Note: IGF-1 values are reported as mean values calculated from 4 animals, adjusted by baseline, i.e., values minus baseline. The baseline is the average of the values at t-144, -120, -96, -72, -48, -24, and 0 hours.

Example 25

Pharmacodynamic studies of human growth hormone administered by single or daily subcutaneous injections to pituitary-depleted male rats. The objective of this study was to compare the efficacy of different hGH formulations when hypophysectomized male Wistar rats were administered subcutaneously once or for seven consecutive days per day. The study design is as follows:

TABLE 23 study design-sample description

Group # or test Compounds	Sample (I)^a	Description of the samples
Group # or test Compounds	Sample (I)^a	Description of the samples	1	Daily soluble medium-pseudoprolapses	(without hGH)16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH₂PO₄(pH6.5)
2	Low daily soluble Medium-dosage	(without hGH)16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH₂PO₄(pH6.5)	1	Daily soluble medium-pseudoprolapses
2	Low daily soluble Medium-dosage		3	Daily soluble Medium-high dose	(without hGH)16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH₂PO₄(pH6.5)
4	Daily soluble-low dose	0.71mg/ml rhGH, 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH₂PO₄(pH6.5)	3	Daily soluble Medium-high dose
4	Daily soluble-low dose		5	Each timeSoluble-high dose	1.0mg/ml rhGH, 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH₂PO₄(pH6.5)
6	Soluble single bolus high dose	3.5mg/ml rhGH, 16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH₂PO₄(pH6.5)	5	Each timeSoluble-high dose
6	Soluble single bolus high dose		7	Polyarginine crystals-high dose	18.7mg/ml rHGH crystals, 250mM NaOAc, 6% PEG-6000, 25mM Tris-HCl (pH8.6), 3.6mg/ml polyarginine HCl (rhGH: polyarginine in a molar ratio of 1: 0.587)

8	Vehicle control-protamine crystals	250mM Na0Ac, 6% PEG-6000, 25mM Tris-HCl (pH8.6), 0.75mg/ml protamine sulfate
8	Vehicle control-protamine crystals		9	Protamine crystals-low dosage	3.3mg/ml rHGH crystals, 250mM NaOAc, 6% PEG-6000, 25mM Tris-HCl (pH8.6), 0.75mg/ml protamine sulfateProtein (rhGH polyarginine molar ratio 1: 1.715)
10	Protamine crystals-high dose	18.7mg/ml rHGH crystals, 250mM NaOAc, 6% PEG-6000, 25mM Tris-HCl (pH8.6), 4mg/ml protamine sulfate (molar ratio rhGH: polyarginine 1: 1.715)	9	Protamine crystals-low dosage
10	Protamine crystals-high dose		11	Medium control-poly arginine crystals	250mM NaOAc, 6% PEG-6000, 25mM Tris-HCl (pH8.6), 3.6mg/ml polyarginine-HCl
12	Vehicle control-Single bolus injection	16.7mg/ml D-mannitol, 26.7mg/ml sucrose, 50mM NaH₂PO₄(pH6.5)	11	Medium control-poly arginine crystals

^aAll samples were prepared under sterile conditions using WFI. After adjusting the solutions to their respective final volumes, the carrier and soluble hGH samples were filtered with a 0.22 μm filter.

TABLE 24 study design-dosing

Group # or test Compounds	Administration level (mg/kg)	Administration concentration (mg/ml)	Volume of administration (μ l)	Dosing regimens	Number of animals (Male)
Group # or test Compounds	Administration level (mg/kg)	Administration concentration (mg/ml)	Volume of administration (μ l)	Dosing regimens	Number of animals (Male)	1	0	0	200	7 administration daily	13
2	0	0	20	7 administration daily	11	1	0	0	200	7 administration daily	13
2	0	0	20	7 administration daily	11	3	0	0	80	7 administration daily	11
4	0.143	0.71	20	7 administration daily	11	3	0	0	80	7 administration daily	11
4	0.143	0.71	20	7 administration daily	11	5	0.8	1	80	7 administration daily	12
6	5.6	3.5	160	Day 1	11	5	0.8	1	80	7 administration daily	12
6	5.6	3.5	160	Day 1	11	7	5.6	18.7	30	Day 1	12
8	0	0	30	Day 1	11	7	5.6	18.7	30	Day 1	12
8	0	0	30	Day 1	11	9	1	3.3	30	Day 1	12
10	5.6	18.7	30	Day 1	12	9	1	3.3	30	Day 1	12
10	5.6	18.7	30	Day 1	12	11	0	0	30	Day 1	11
12	0	0	30	Day 1	11	11	0	0	30	Day 1	11

Upon arrival, 138 female Wistar rats, weighing approximately 90-100 grams and approximately 25-30 days old, were housed under controlled conditions (approximately 23 ± 3 ℃ temperature, 30-70% relative humidity, 12 hours light and 12 hours dark in each 24 hour cycle, 10-15 air exchanges per hour) and were allowed free access to pure water and laboratory grain throughout the study. Rats were allowed to acclimate for two weeks prior to testing.

138 rat samples were given according to the concentrations, volumes and dosing schedule in Table 24. Test compounds were administered subcutaneously in the back in a single bolus injection once or once daily for seven consecutive days. Injection sites were shaved and marked up to three days prior to dosing, after which time injection was facilitated as needed. Test compounds were administered using a 30-gauge x 8mm needle attached to a 300 μ l syringe. Before being drawn into the syringe and again before administration, the test compound was carefully inverted to ensure suspension or solution homogeneity without causing foaming.

Body weight gain was measured and recorded twice weekly between-3 weeks and-2 weeks and daily from-7 days to 14 days. When administered, the rat body weight was approximately 100 g. + -. 10%. The percentage results of induced growth are provided in figures 22 and 23 and summarized in tables 25 and 26. In Table 25, "high dose" means 5.6 mg/kg/week. The data show a comparison of the weight gain of rats during the same seven days of single injection of rhGH polyarginine (group 7, examples 18 and 19) or rhGH protamine (groups 9 and 10, examples 18 and 19) crystals versus daily injection of either control (group 1, no hGH) or soluble hGH samples (groups 4 and 5). Group 1, pseudoprolapsed rats, showed normal growth over a seven day period. Furthermore, rats given rhGH polyarginine once a seven day injection (group 7) had a higher percentage of induced growth than those given soluble hGH daily for seven days (group 5). These results demonstrate that hGH crystals and formulations according to the invention are as effective as soluble rhGH administered daily for a week.

TABLE 25 weight gain induced in hypophysectomized rats for 8 days

TABLE 26 daily induced weight gain in hypophysectomized rats

Example 26

hGH was crystallized from sodium acetate and protamine sulfate. Here, a frozen bulk feed solution of soluble recombinantly produced hGH (rhGH) was obtained from two stocks, one derived from E.coli (Novartis) and the other from yeast (Lucky Gold). Independent analysis of rhGH from e.coli and yeast stock solutions, regardless of its source, resulted in rhGH with identical crystallization and solubility characteristics. Approximately 3.3ml (10-20mg/ml) of thawed rhGH feed solution was purified using a 10DG desalting column supplied by BioRad. Before sample loading, the column was treated by washing with 30ml Tris-HCl (10mM, pH 8.0). The rhGH sample was then loaded and allowed to enter the column by gravity. After discarding the first 3ml eluate, another 5.0ml 10mM Tris-HCl pH8.0 was added. 4.5ml of desalted rhGH was eluted and collected. Concentration was then performed by centrifugation using a Millipore concentrator (MWCO10,000) at 3500rpm for 20-30 minutes. The concentration of hGH was in the range of 30mg/ml as measured by absorbance at 280nm/0.813(1mg/ml hGH a280 ═ 0.813 absorbance units). Crystals were produced by adding deionized water, Tris-HCl (pH8.6), PEG-4000, protamine sulfate and sodium acetate to final concentrations of 100mM, 6% (v/v), 2mg/ml and 500mM, respectively, in total solution, to a final protein concentration of 15 mg/ml. The solution was then gently mixed and incubated at 33 ℃ for 12-16 hours. Needle-shaped crystals having a length of about 2 to 25 μm were obtained. After centrifugation and pelleting of the crystals, the supernatant was extracted and the crystallization yield was measured to be greater than 90%.

Example 27

hGH was crystallized from sodium acetate and polyarginine HCl. Here, a frozen bulk feed solution of soluble recombinantly produced hGH (rhGH) was obtained from two stocks, one derived from E.coli (Novartis) and the other from yeast (Lucky Gold). Independent analysis of rhGH from e.coli and yeast stock solutions, regardless of its source, resulted in rhGH with identical crystallization and solubility characteristics. Approximately 3.3ml (10-20mg/ml) of thawed rhGH feed solution was purified using a 10DG desalting column supplied by BioRad. Before sample loading, the column was treated by washing with 30ml Tris-HCl (10mM, pH 8.0). The rhGH sample was then loaded and allowed to enter the column by gravity. After discarding the first 3ml eluate, another 5.0ml 10mM Tris-HCl pH8.0 was added. 4.5ml of desalted rhGH was eluted and collected. Concentration was then performed by centrifugation using a Millipore concentrator (MWCO10,000) at 3500rpm for 20-30 minutes. The concentration of hGH was in the range of 30mg/ml as measured by absorbance at 280nm/0.813(1mg/ml hGH a280 ═ 0.813 absorbance units). Crystals were generated by adding deionized water, Tris-HCl (pH8.6), PEG-4000, polyarginine HCl and sodium acetate to final concentrations of 100mM, 2% (v/v), 2mg/ml and 500mM, respectively, in total solution, with a final protein concentration of 15 mg/ml. The solution was then gently mixed and incubated at 33 ℃ for 12-16 hours. Needle-shaped crystals having a length of about 2 to 25 μm were obtained. After centrifugation and pelleting of the crystals, the supernatant was extracted and the crystallization yield was measured to be greater than 90%.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the disclosure (including the appended embodiments).

Claims

1. Calcium crystals of human growth hormone or a human growth hormone derivative.

2. Monovalent cation crystals of human growth hormone or a human growth hormone derivative.

3. Protamine crystals of human growth hormone or a human growth hormone derivative.

4. A crystal of human growth hormone or a human growth hormone derivative of polyarginine.

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

6. The monovalent cation crystal according to claim 5, wherein said monovalent cation is sodium.

7. The crystal according to any one of claims 1, 2, 3 or 4, wherein a single administration of said crystal to a mammal provides an in vivo hGH serum concentration in said mammal selected from the group consisting of:

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

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

(c) between about 1ng/ml to about 100ng/ml hGH, over a period of time selected from the group consisting of:

(i) between about 0.5 hours and about 40 days after administration;

(ii) between about 0.5 hours and about 10 days after administration;

(iii) between about 0.5 hours and about 7 days after administration; and

(iv) between about 0.5 hours and about 1 day after administration.

8. The crystal according to any one of claims 1, 2, 3 or 4, wherein a single administration of said crystal to a mammal provides an in vivo IGF-1 serum elevation in said mammal which is above the baseline IGF-1 level prior to said administration selected from the group consisting of:

(a) between about 5ng/ml to about 2,500 ng/ml; and

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

passing a time period selected from the group consisting of:

(i) between about 0.5 hours and about 40 days after administration;

(ii) between about 0.5 hours and about 7 days after administration.

9. A crystal according to any of claims 1, 2, 3 or 4, wherein said crystal has a relative bioavailability of at least 50% or more compared to the relative bioavailability of the same dose of soluble hGH delivered by the same route of administration, wherein said bioavailability is measured by AUC of the serum concentration of hGH within said soluble hGH and said crystal as a whole.

10. The crystal according to claim 7 or 8, wherein the mammal is a human.

11. The crystal according to claim 1, wherein said crystal comprises from about 1 to about 500 calcium molecules per monomer of human growth hormone or human growth hormone derivative.

12. The crystal according to claim 2, wherein said crystal comprises from about 1 to about 500 monovalent cations per monomer of human growth hormone or a human growth hormone derivative.

13. The crystal according to claim 1, wherein the crystal comprises a calcium salt selected from the group consisting of calcium acetate, calcium chloride, calcium sulfate and calcium gluconate.

14. The crystal according to claim 13, wherein the calcium salt is calcium acetate.

15. The crystal according to claim 6, wherein the crystal comprises a sodium salt selected from the group consisting of sodium citrate, sodium phosphate and sodium acetate.

16. The crystal according to claim 15, wherein the sodium salt is sodium acetate.

17. A composition comprising crystals of human growth hormone or a human growth hormone derivative and an excipient, wherein the crystals are selected from the group consisting of:

(a) calcium crystals of human growth hormone or a human growth hormone derivative;

(b) monovalent cation crystals of human growth hormone or a human growth hormone derivative;

(c) protamine crystals of human growth hormone or a human growth hormone derivative;

(d) a crystal of human growth hormone or a human growth hormone derivative of polyarginine.

18. A composition according to claim 17 wherein said crystals and said excipient are present in said composition in a molar ratio of hGH to excipient of from about 1: 10 to about 1: 0.125.

19. The composition according to claim 17, wherein the excipient is selected from the group consisting of amino acids, salts, alcohols, carbohydrates, proteins, lipids, surfactants, polymers, polyamino acids, and mixtures thereof.

20. The composition according to claim 19, wherein the excipient is selected from the group consisting of protamine, polyvinyl alcohol, cyclodextrin, dextran, calcium gluconate, polyamino acids, polyethylene glycol, dendrimers, polyornithine, polyethyleneimine, chitosan, and mixtures thereof.

21. The composition according to claim 20, wherein the excipient is selected from the group consisting of protamine, polyarginine, polyethylene glycol, and mixtures thereof.

22. The composition according to claim 17, wherein the concentration of human growth hormone or human growth hormone derivative in said composition is selected from the group consisting of:

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

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

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

23. A method of treating a mammal having a disorder associated with human growth hormone deficiency or ameliorated by treatment with human growth hormone, which comprises the step of administering to said mammal a therapeutically effective amount of the crystal of any one of claims 1, 2, 3 or 4 or the composition of claim 17.

24. A method of inducing weight gain in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of the crystal of any one of claims 1, 2, 3 or 4 or the composition of claim 17.

25. A method according to claim 24, wherein said mammal is a hypophysectomized rat and the weight gain induced in said rat following administration of said crystal by once a week injection is between 5% and about 40%.

26. A method according to claim 23, wherein said disorder is selected from the group consisting of adult growth hormone deficiency, childhood growth hormone deficiency, prader-willi syndrome, turner's syndrome, short bowel syndrome, chronic renal insufficiency, idiopathic short stature, dwarfism, hypopituitarism dwarfism, bone regeneration, female infertility, intrauterine growth retardation, AIDS-related cachexia, crohn's disease, and burns.

27. The method according to claim 26, wherein said disorder is pediatric growth hormone deficiency and said method results in an annual growth rate in said mammal of between about 7 and about 11 cm.

28. The method according to claim 23 or 24, wherein said crystal or composition is administered to said mammal by an oral route, a parenteral route, a subcutaneous route, or an intramuscular route.

29. A method according to claim 28, wherein said crystal or composition is administered to said mammal by the subcutaneous route using a needle having a gauge greater than or equal to 27.

30. The method according to claim 23 or 24, wherein said crystal or composition is administered to said mammal by needle-free injection or variable dose infusion pump.

31. The method according to claim 23 or 24, wherein the crystal or composition is administered to the mammal by a time regimen selected from the group consisting of:

(a) about once every three days;

(b) about once a week;

(c) about once every two weeks; and

(d) about once a month.

32. A method according to claim 23 or 24, wherein the mammal is a human.

33. A method for producing calcium crystals, monovalent cation crystals, protamine crystals or poly-arginine crystals of human growth hormone or a human growth hormone derivative, the method comprising the steps of:

(a) mixing a solution of human growth hormone or a human growth hormone derivative with a crystallization solution comprising a calcium salt or a monovalent cation crystalline salt and an ionic polymer, wherein the ionic polymer is protamine or polyarginine; and

(b) incubating the crystal solution at a temperature between about 4 ℃ and about 37 ℃ for greater than about 12 hours until calcium crystals, monovalent cation crystals, protamine crystals, or polyarginine crystals of human growth hormone or a human growth hormone derivative are produced.

34. The method according to claim 33, wherein the ionic polymer is polylysine.

35. The method according to claim 33, wherein the ionic polymer is a mixture of any two or more of protamine, polyarginine and polylysine.

36. A method for producing calcium crystals or monovalent cation crystals of human growth hormone or a human growth hormone derivative, the method comprising the steps of:

(a) mixing a solution of human growth hormone or a human growth hormone derivative with a crystallization buffer to produce a crystallization solution;

(b) adding deionized water to the crystallization solution;

(c) adding a precipitating agent to the crystallization solution;

(d) adding a calcium salt or a monovalent cation salt to the crystallization solution;

(e) incubating the crystallization solution at a temperature between about 10 ℃ and about 40 ℃ for about 2 to about 168 hours until calcium crystals or monovalent cation crystals of human growth hormone or a human growth hormone derivative are formed; and

(f) adding an ionic polymer or ionic small molecule to the calcium crystals or monovalent cation crystals of the human growth hormone or human growth hormone derivative.

37. A method for producing calcium crystals or monovalent cation crystals of human growth hormone or a human growth hormone derivative, the method comprising the steps of:

(b) adding deionized water to the crystallization solution;

(c) adding an ionic small molecule or ionic polymer to the crystallization solution;

(e) incubating the crystallization solution at a temperature between about 10 ℃ and about 40 ℃ for about 2 to about 168 hours until calcium crystals or monovalent cation crystals of human growth hormone or a human growth hormone derivative are formed.

38. The method according to claim 37, wherein after step (b) and before step (c), said method comprises the step of adding a precipitating agent to said crystallization solution.

39. The method according to any one of claims 33, 36 or 37, wherein said calcium salt is selected from the group consisting of calcium acetate, calcium chloride, calcium gluconate, and calcium sulfate.

40. The method according to claim 39, wherein the calcium salt is calcium acetate.

41. The method according to any one of claims 33, 36 or 37, wherein said monovalent cation is selected from the group consisting of lithium, sodium, potassium and ammonium.

42. The method according to claim 41, wherein said monovalent cation is sodium.

43. The method according to any one of claims 33, 36 or 37, wherein said monovalent cation salt is selected from the group consisting of sodium citrate, sodium phosphate and sodium acetate.

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

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

46. The method according to claim 45, wherein the pH buffer has a pH selected from the group consisting of:

(a) a pH between about pH6 and about pH 10;

(b) a pH between about pH7 and about pH 10;

(c) a pH between about pH6 and about pH 9; and

(d) a pH between about pH7.8 and about pH 8.9.

47. The method according to claim 45, wherein the pH buffer is a buffer selected from the group consisting of Tris, HEPES, acetate, phosphate, citrate, borate, imidazole and glycine.

48. A process according to claim 36 or 38, wherein the precipitating agent is a non-ionic small molecule or a non-ionic polymer.

49. The method according to claim 48, wherein said non-ionic polymer is selected from the group consisting of polyethylene glycol, polyvinyl alcohol, and mixtures thereof.

50. The method according to claim 49, wherein said polyethylene glycol has a molecular weight selected from the group consisting of:

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

(b) a molecular weight of about 6000;

(c) a molecular weight of about 4000; and

(d) a molecular weight of about 3350.

51. The method according to claim 50, wherein said polyethylene glycol is present in said crystallization solution at a concentration between about 0.5% and about 20% (w/v).

52. The method according to claim 36 or 38, wherein the precipitating agent is selected from the group consisting of amino acids, peptides, polyamino acids, and mixtures thereof.

53. The method according to claim 33, 36 or 37, wherein said human growth hormone or human growth hormone derivative is present in said crystallization solution in a concentration selected from the group consisting of:

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

(b) a concentration of between about 2mg/ml and about 50 mg/ml; and

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

54. The method according to claim 33, 36 or 37, wherein said calcium salt or said monovalent cation salt is present in said crystallization solution at a concentration selected from the group consisting of:

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

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

55. The method according to claim 33, 36 or 37, wherein said crystallization solution is incubated for a time and at a temperature selected from the group consisting of:

(a) between about 0.25 days and about two days at a temperature of about 33 ℃;

(b) between about 0.25 days and about two days at a temperature of about 25 ℃; and

(c) between about 0.25 days and about two days at a temperature of about 15 ℃.

56. The method according to claim 36 or 37, wherein the ionic small molecule is selected from the group consisting of amino acids, peptides and mixtures thereof.

57. A method according to claim 36 or 37, wherein the ionic polymer is selected from the group consisting of protamine, polysaccharide, polyamino acid, polyarginine, polylysine, polyglutamic acid, dendrimer, poly (t-amino acid), poly (l-amino acid), poly,

Polyornithine, polyethyleneimine, chitosan, and mixtures thereof.

58. The method according to claim 57, wherein the ionic polymer is protamine or polyarginine.

59. The process according to claim 36 or 37, wherein, in step (e) of claim 36 or step (e) of claim 37, said crystallization solution is incubated at a temperature between about 4 ℃ and about 40 ℃ for about 4 to about 48 hours until calcium crystals or monovalent cation crystals of human growth hormone or a human growth hormone derivative are formed.

60. The method according to claim 36 or 37, wherein, in step (e) of claim 36 or step (e) of claim 37, said human growth hormone or human growth hormone derivative is present in said crystallization solution at a concentration of between about 2mg/ml and about 100 mg/ml.

61. The method according to claim 36 or 37, wherein, in step (e) of claim 36 or step (e) of claim 37, said human growth hormone or human growth hormone derivative is present in said crystallization solution at a concentration of between about 14.5mg/ml and about 15.5 mg/ml.

62. The method according to claim 36 or 37, wherein the crystallization buffer is selected from the group consisting of Tris-HCl buffer, glycine buffer, HEPES buffer, imidazole buffer, Bis-Tris buffer, AMP, AMPD, AMPSO, bicine, ethanolamine, glycylcycline, TAPS, taurine, Triane, and mixtures thereof.

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

64. The method according to claim 36 or 37, wherein in step (a) the crystallization buffer has a pH selected from the group consisting of:

(a) a pH between about 3 and about 10;

(b) a pH between 6 and about 9; and

(c) a pH between about 7.5 and about 10.

65. The method according to claim 36 or 37, wherein, in step (e) of claim 36 or step (e) of claim 37, said crystallization buffer in said crystallization solution brings said solution to a pH selected from the group consisting of:

(a) a pH between about 3 and about 10;

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

(c) a pH between about 7.5 and about 9.5.

66. The method according to claim 64 or 65, wherein the buffer is selected from the group consisting of Tris, HEPES, acetate, phosphate, citrate, borate, imidazole and glycine.

67. The method according to claim 40, wherein said calcium acetate is in the form of an aqueous solution having a pH selected from the group consisting of:

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

(b) a pH between 7.0 and about 8.6.

68. The method according to claim 40, wherein, in step (e) of claim 36 or step (e) of claim 37, said calcium acetate is present in said crystallization solution at a concentration selected from the group consisting of:

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

(b) a concentration of between about 85mM and about 100 mM.

69. The method according to claim 44, wherein said calcium acetate is in the form of an aqueous solution having a pH selected from the group consisting of:

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

(b) a pH between 7.0 and about 8.6.

70. The method according to claim 45, wherein, in step (e) of claim 36 or step (e) of claim 37, said calcium acetate is present in said solution at a concentration selected from the group consisting of:

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

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

71. The method according to claim 36 or 37, wherein, in step (e) of claim 36 or step (e) of claim 37, the solution is incubated at a temperature between about 4 ℃ and about 37 ℃ for about one day to about two days.