WO2004024753A1

WO2004024753A1 - Process for manufacturing crystals of growth hormone

Info

Publication number: WO2004024753A1
Application number: PCT/SE2003/001422
Authority: WO
Inventors: Jonas Fransson; Mitra Mosharraf; Leena Lehtikari; Sinikka Uotila; Kalevi Visuri
Original assignee: Pharmacia AB; Pfizer Health AB
Current assignee: Pfizer Health AB
Priority date: 2002-09-13
Filing date: 2003-09-12
Publication date: 2004-03-25
Anticipated expiration: 2005-03-13
Also published as: CA2498525A1; EP1539797A1; MXPA05002826A; JP2006513986A; AU2003259002A1; SE0202719D0; BR0314187A

Abstract

A process is provided for manufacturing crystals of growth hormone (GH), or functional derivatives thereof, comprising the following steps : (a) preparing an aqueous solution of GH, or functional derivatives thereof, comprising 2-propanol, (b) incubating the solution prepared in step (a) to crystallize said GH, or functional derivatives thereof, and (c) isolating the crystals formed in step (b). A novel use of 2-propanol as a crystallizing agent for manufacturing crystals of GH, or functional derivatives thereof is also provided. In addition, crystals obtainable by the process and pharmaceutical compositions comprising the crystals are provided. The crystals are useful as a medicament, for the manufacture of such a medicament, and in a method of treating a mammal, including man.

Description

PROCESS FOR MANUFACTURING CRYSTALS OF GROWTH HORMONE

The present invention relates to a novel process for manufacturing crystals of growth hormone (GH) or functional derivatives thereof. It also relates to crystals of growth hormone obtainable by said process and composi- tions containing such crystals.

BACKGROUND TO THE INVENTION

Growth hormone refers in general to animal, including human, growth hormone, such as human growth hormone (hGH) , bovine growth hormone (bGH) , fish growth hormone and porcine growth hormone (pGH) . hGH is a protein consisting of a single chain of 191 amino acids. The molecule is cross-linked by two disul- phide bridges, and the monomeric form has a molecular weight of 22 kDa. However, pituitary human growth hormone is not homogeneous. For example, a smaller 20 kDa hGH variant produced from the same gene is also known. The "basic hGH" variant (hGH-V) expressed by the placenta during pregnancy is another analogue which is a product of a separate gene. Like the 22 kDa hGH, it consists of 191 amino acids, but in various positions throughout the molecule, 13 of the amino acid residues are different. See e g Bewley T A et al , Adv Enzymol 42:73-166, 1975, and Frankenne F et al , J Clin Endocrin and Metabol 66:1171-80, 1988.

Recombinant hGH (22 kDa) has been commercially available for several years. It is preferred over the pituitary derived products, since the product prepared from human tissue might contain infectious agents, such as the causative agent of Creutzfeldt-Jacob' s disease.

There are therapeutically useful recombinant hGH preparations present on the market: the authentic one, e g Genotropin^®, Pharmacia, and an analogue with an addi- tional methionine residue at the N-terminal end, e g Somatonorm^® . hGH is used to stimulate linear growth in patients with hypopituitary dwarfism or Turner's syndrome, but other indications have also been suggested.

The stability of proteins is generally a problem in the pharmaceutical industry. It has often been solved by drying the protein in different drying processes, such as freeze-drying. The protein has thereafter been distrib- uted and stored in dried form. The patient necessarily has to reconstitute the dried protein in a solvent before use, which is a disadvantage and of course is an inconvenience for the patient. The freeze-drying process is a costly and time-consuming process step. For a patient who needs daily injections of a growth hormone, e g hGH, and especially when the patient is a child, it is of importance that the product is easy to handle, to dose and inject. The reconstitution of freeze-dried hGH demands prudence and carefulness and should preferably be avoided. Another way of manufacturing growth hormone is by crystallization. Crystals of growth hormone can be used for various new formulations of the hormone, e g in- jectable suspensions, implants and topical formulations of various types . Various attempts to obtain suitable crystals of GH have been made. Jones et al , Bio/Technology 5:499-500, 1987, crystallized recombinant human growth hormone, using the hanging drop technique, from a solution containing polyethylene glycol and β-octyl glucoside. Borisova et al, Doklady Biochemistry 301/1-6:207-

210, 1988, crystallized human Des-Phen^somatotropin, but not hGH, using the hanging drop technique, in acetone with additives.

Clarkson et al , J Mol Biol 208:719-721, 1989, re- ports of various crystallization methods. These include a hanging drop technique using ethanol, methanol or acetone in the buffer, and a batchwise technique using paralde- hyde . ..

In US 5 780 599 to Novo Nordisk A/S, a method of producing chemically stable and biologically active growth hormone cation crystals is disclosed. The method comprises the steps: addition of divalent inorganic cations to a solution of GH at a pH between 5 and 8, growing of crystals at a temperature of 0°C-30°C, and isolation of the crystals. The obtained crystals always in- elude a divalent inorganic cation. The preferred divalent inorganic cation is Zn²⁺. Preferably, an organic solvent is added together with the cation. While the experiments are concerned with the organic solvents acetone and etha- nol, it is briefly proposed that 2-propanol is also a suitable organic solvent in conjunction with divalent inorganic cations.

In WO 94/10192 to Kabi Pharmacia AB, a process for manufacturing crystals of GH is disclosed, involving the use of an aromatic alcohol, such as benzyl alcohol. There is a demand on the market for better ways of administering hGH. Crystals of hGH can be used in a suspension or in an aqueous injectable solution together with buffers, and with or without preservatives. They can also be used in depot formulation, as e g an oily or aqueous suspension, or as an implant, and thus give a slow release of the medicament. If the crystals are large enough, they can be used as a powder and e g be spread on the surface of a wound.

Hence, there is a need for novel and reproducible ways of obtaining crystals of GH at industrial scale. A novel and inventive process for production of crystals of GH is presented in this patent application. SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel process for manufacturing crystals of growth hormone (GH) , or functional derivatives thereof. It is also an object of the present invention to provide such a process, which is robust and technically simple to perform.

It is another object of the present invention to provide such a process, which employs pharmaceutically acceptable conditions.

It is an object of the present invention to provide such a process, which has a high yield.

For these and other purposes that will be obvious from the following description, the present invention provides in its broadest aspect a novel process for manufacturing crystals of growth hormone (GH) , or functional derivatives thereof, comprising the following steps: (a) preparing an aqueous solution of GH, or functional derivatives thereof, comprising 2-propanol, (b) incubat- ing the solution prepared in step (a) to crystallize said GH, or functional derivatives thereof, and (c) isolating the crystals formed in step (b) .

In a particular process according to the invention, said 2-propanol is present in a concentration of at most 25% v/v. Preferably, said 2-propanol is present in a concentration in the range of 5-25% v/v, more preferably in the range of 16-20% v/v, even more preferably in the range of 18-20% v/v.

In one process according to the invention, the solu- tion prepared in step (a) also comprises a buffer selected from sodium/potassium phosphate, citric acid, a combination of sodium/potassium phosphate and citric acid, and BisTris-HCl. In a preferred process according to the invention, said buffer is sodium/potassium phos- phate.

It is preferred that the buffer is present in a concentration of more than 10 mM. It is particularly pre- ferred that the buffer is present in a concentration range of 10 mM-0.8 M, preferably 20 mM-0.8 M, more preferably 20 mM-O.l M.

In a particular process according to the invention, the solution prepared in step (a) is adjusted to a pH of less than about 6.2. In a preferred process according to the invention, the solution prepared in step (a) is adjusted to a pH in the range of 5.5-6.2, preferably in the range of 5.5-5.7. In another process according to the invention, the solution prepared in step (a) is adjusted to a pH of more than about 7.0, preferably in the range of 7.0-9.0.

In one process according to the invention, said GH, or functional derivatives thereof, is present in the so- lution prepared in step (a) in a concentration above 0.1 mg/ml, preferably above 1 mg/ml . In a preferred process according to the invention, said concentration of GH, or functional derivatives thereof, is in the range of 0.1-20 mg/ml, preferably 1-20 mg/ml, more preferably in the range of 12-18 mg/ml.

In a particular process according to the invention, said GH is human GH.

In a particular process according to the invention, the molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, is less than 0.5, and the molar ratio of zinc ions to GH, or functional derivatives thereof, is less than 0.003 in said solution of step (a) .

In a preferred process according to the invention, the molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, is less than 0.2, preferably less than 0.1, more preferably less than 0.003.

In a particular process according to the invention, the concentration of divalent inorganic cations is lower than about 0.3 mM, preferably lower than about 0.1 mM, in said solution of step (a) . In a preferred process according to the invention, said solution of step (a) is substantially void of divalent inorganic cations. In one process according to the invention, the concentration of zinc ions is lower than about 0.002 mM in said solution of step (a) . In a preferred process according to the invention, said solution of step (a) is sub- stantially void of zinc ions.

Moreover, the present invention provides a novel use of 2-propanol as a crystallizing agent for manufacturing crystals of GH, or functional derivatives thereof.

It also provides use of 2-propanol in an aqueous so- lution comprising growth hormone (GH) , or functional derivatives thereof, for manufacturing crystals of GH, or functional derivatives thereof, from said aqueous solution. In a preferred use according to the invention, said solution has a molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, of less than 0.5 and a molar ratio of zinc ions to GH, or functional derivatives thereof, of less than 0.003.

The present invention also provides crystals of GH, or functional derivatives thereof, obtainable by the process according to the invention.

In addition, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of crystals of growth hormone (GH) , or functional derivatives thereof, according to the inven- tion, and a suitable pharmaceutical carrier therefor. In a preferred composition according to the invention, said composition is a suspension for injection. In another preferred composition according to the invention, said composition is a depot formulation. In yet another pre- ferred composition according to the invention, said composition is a dry formulation.

The present invention provides crystals of growth hormone (GH) , or functional derivatives thereof, according to the invention, for use as a medicament. The present invention also provides a novel use of crystals of growth hormone (GH) , or functional derivatives thereof, according to the invention, for the manu- facture of a medicament for treating a mammal, including man, in need of GH, or functional derivatives thereof.

Finally, the present invention provides a method of treating a mammal, including man, in need of growth hor- mone (GH) , or functional derivatives thereof, comprising administering to said mammal, in need of such a treatment, a therapeutically effective amount of crystals of GH, or functional derivatives thereof, according the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Fig 1 is a diagram showing the effect of 2-propanol concentration on the solubility of hGH crystals.

Fig 2 is a diagram showing the effect of phosphate concentration on the solubility of hGH crystals in 2-propanol .

Fig 3 is a diagram showing the effect of pH on the solubility of hGH crystals in 2-propanol.

Fig 4 is a diagram showing the effect of pH on the solubility of hGH crystals in phosphate combined with 2-propanol .

Fig 5 is a diagram showing the effect of temperature on the solubility of hGH crystals in 2-propanol.

Fig 6 is a diagram showing the effect of temperature on the solubility of hGH crystals in phosphate combined with 10% (v/v) 2-propanol.

Fig 7 is a diagram showing the effect of temperature on the solubility of hGH crystals in phosphate combined with 20% (v/v) 2-propanol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is generally based on the surprising finding that 2-propanol is a particularly suitable crystallizing agent for crystallization of GH, or functional derivatives thereof, from a solution. The terms "growth hormone" and "GH" refer in general to animal growth hormone, including human growth hormone. The GH may be derived either from natural sources or by recombinant sources. It is preferred that the GH is a re- combinant GH (rGH) . Examples of GH include human growth hormone (hGH) , bovine growth hormone (bGH) , fish growth hormone, including GH from salmon, trout and tuna, porcine growth hormone (pGH) , and ovine growth hormone. It is preferred that the GH is hGH. The term "functional derivatives" of growth hormone is hereby meant to include both naturally occurring and engineered analogues or variants. Examples of naturally occurring analogues or variants include the 20 kDa hGH variant produced from the same gene, and the "basic hGH" variant (hGH-V) expressed by the placenta during pregnancy is another analogue which is a product of a separate gene. Engineered analogues or variants include GH mutants produced by genetic engineering and recombinant technology as well as chemically or enzymatically modi- fied GH.

The term "crystallization" refers to a well-known phenomenon occurring in supersaturated solutions of molecules, whereby crystals are formed by aggregation of molecules in an ordered, repetitive fashion. When a more random aggregation of molecules occurs, amorphous precipitates are obtained. Specifically, crystallization of proteins, such as GH, takes place in supersaturated solutions of GH, i e solutions wherein the concentration of GH is brought above the saturation point. This can be achieved in a number of ways, e g lowering of the temperature of the solvent or addition of a crystallizing agent to the solvent .

In general terms, crystallization of proteins is useful as a method of purification, a confirmation of protein homogeneity, a method of stable storage, and as a starting point for determination of the three-dimensional structure of the protein through X-ray crystallography. Due to the large size of proteins, crystals tend to grow rather slowly, especially if it is desirable to obtain large crystals.

The particular crystallization method employed is governed by a number of factors, including the nature of the solvent and the crystallizing agents, and the amount of protein available. In most cases, the most suitable method and specific conditions are not obvious from the current theoretical knowledge, but must be experimentally confirmed and optimized.

If large amounts of protein are available, crystallization can be performed batchwise. Briefly, a suitable crystallizing agent is added to a solution of the desired protein, whereby a supersaturated solution is achieved. Crystallization can also be achieved through a more gradual change in solvent conditions by increasing the concentration of the crystallizing agent over a period of minutes, hours, or days. The two basic ways of achieving this are through dialysis-based systems and diffusion- based systems.

In dialysis-based systems, a dialysis membrane separates a solution of protein and crystallizing agent from a solution of crystallizing agent, having higher concentration of crystallizing agent than the protein . solution. Through dialysis, the protein solution becomes supersaturated.

In diffusion-based systems, such as the "hanging drop" method and the "sitting drop" method, the protein is brought to a state of supersaturation through vapor diffusion of volatile species between the sample, containing the protein in question, and a reservoir. The diffusion continues until the vapor pressures in the sample and in the reservoir are equal .

In the "hanging drop" method, a protein sample drop, typically 5-30 μl , is placed on a cover slide. The slide is placed on the top of a chamber containing a reservoir reagent solution, such that the slide, from which the drop is hanging, constitutes the ceiling of the chamber. The chamber is well sealed. The reagent solution typically comprises the crystallizing agent and non-volatile species, such as salts and buffers, and it is critical that the drop also contains some of the non-volatile species. In order to achieve equilibrium, volatile species evaporate from the drop until the vapor pressure in the drop and the chamber are equal , leading to an increased concentration of protein and non-volatile species in the drop .

Typically, a small volume, such as 1-30 μl , of protein solution is mixed with a small volume, such as 1-30 μl, of reagent solution, which is identical to the reservoir solution. Equilibrium is reached when the reagent concentration in the drop is nearly the same as that in the chamber. Thus, the final volume of the drop is approximately the same as the initial volume of reagent solution in the drop.

The principle of the "sitting drop" method is the same as of "hanging drop" method, with the exception that the drop is placed on a drop holder instead of a cover glass. If more crystals are desirable, this method allows for use of larger volumes than the "hanging drop" method. 2-propanol, also known as isopropyl alcohol, is a colorless fluid, which is miscible with water and most organic fluids. 2-propanol is a pharmaceutically acceptable solvent. It is volatile, and can therefore be removed from the product with simple drying procedures. Finally, 2-propanol is inexpensive and user-friendly. The term "reagent" refers generally to any chemical species used in the crystallization process, including buffers, salts, and crystallizing agents. The term "crystallizing agent" refers to the particular component which causes the crystallization.

An embodiment of the invention provides a process for manufacturing crystals of growth hormone (GH) , or functional derivatives thereof, comprising the following steps :

(a) preparing an aqueous solution of GH, or functional derivatives thereof, comprising 2-propanol, (b) incubating the solution prepared in step (a) to crystallize said GH, or functional derivatives thereof, and (c) isolating the crystals formed in step (b) .

The preparing step (a) may involve preparing a batch crystallization, a dialysis-based crystallization, a dif- fusion-based crystallization, such as "hanging drop" crystallization and "sitting drop" crystallization, or any other suitable crystallization method.

Thus, step (a) may simply involve mixing of a suitable volume of a solution comprising 2-propanol with an aqueous solution of GH, or a functional derivative thereof. The resulting solution can be used for batchwise production of crystals. When dialysis- or diffusion-based crystallization methods are employed, step (a) may involve mixing of a suitable volume of the reservoir solu- tion comprising 2-propanol to an aqueous solution of GH, or a functional derivative thereof. Step (a) may also involve adjusting the pH of the solution, e g by including a buffer, or adding other suitable agents to the solution. The incubating step (b) involves allowing crystals to form from the supersaturated solution prepared in step (a) . There are a number of crystallization processes known to a person skilled in the art. On laboratory scale, the "hanging drop" and "sitting drop" methods are commonly preferred. On industrial scale, the most convenient way of incubating the solution in order to obtain crystals is simply to allow the solution to stand for a period of time. Optionally, the incubation may comprise lowering of temperature, gentle stirring or other routine manufacturing procedures.

By way of example, a process according to the invention may be performed in a propeller-stirred vessel under a nitrogen atmosphere. The process is started and kept for 12 h at room temperature. The temperature is then decreased at a linear rate to 6°C in the next 12 h.

The isolating step (c) involves collecting the crys- tals prepared in step (b) in any suitable way. This procedure is well known to the skilled man in the art. The remaining solution may be incubated further in order to obtain more crystals, if possible. The remaining GH in the solution may also be recycled and participate in fur- ther crystallization cycles.

By the process according the invention, crystals of GH, or functional derivatives thereof, are obtainable. Without being limited thereto, the obtained crystals may be rod-shaped, but of varying crystal sizes and more detailed structures. The yield, i e the ratio of crystallized GH to total GH, is typically above 70%, and often as high as 90-95%.

It is contemplated that the aqueous solution of GH, or functional derivatives thereof, is prepared such that it is comprising no more than 25% (v/v) 2-propanol. It is preferred that the 2-propanol concentration is within the range of 5-25% v/v. In order to achieve high yield and/or large crystals, it is preferred that the 2-propanol concentration is within the range of 15-20% or 16-20% v/v, more preferably 18-20% v/v. A particularly preferred 2-propanol concentration is 20% v/v.

In an embodiment of the invention, step (a) of pre- paring the aqueous solution of GH, or functional derivatives thereof, further involves inclusion of a buffer in said solution. The buffer is commonly selected from pharmaceutically acceptable buffers. Examples of suitable buffers are sodium/potassium phosphate, citric acid, a combination of sodium/potassium phosphate and citric acid, and BisTris-HCl. In a preferred embodiment of the invention, the buffer is phosphate buffer. The inclusion of a buffer facilitates maintaining of a suitable pH in the solution throughout the process. The buffer concentration is selected so as to provide a suitable buffering effect without interfering negatively with the crystalli- zation process. Typically, the buffer concentration is above 10 mM. In preferred embodiments, The buffer concentration is in the range of from 10 mM to 0.8 M or from 20 mM to 0.8 M, preferably from 20 mM to 0.1 M.

In another embodiment of the inventive process, the aqueous solution of GH, or functional derivatives thereof, is prepared such that the resulting pH is adjusted to less than about 6.2. Generally, it can be said that the solubility of GH in solutions of water/2 -propanol has a maximum at pH 6.5. From this value, lowering of pH decreases the solubility of GH in the presence of 2-propanol. Thus, in a preferred embodiment, the aqueous solution of GH, or functional derivatives thereof, is prepared such that the resulting pH is adjusted to be within the range of from 5.5 to 6.2, pref- erably from 5.5 to 5.7. It is also contemplated that a pH well- above 6.5 may be employed in the process according to the invention. Accordingly, in a preferred embodiment, the aqueous solution of GH, or functional derivatives thereof, is prepared such that the resulting pH is ad- justed to above 7.0, e g in the range of 7.0-9.0.

In a particularly preferred embodiment, the solution prepared in step (a) is adjusted to a pH of approximately 5.6 with a 0.01-0.7 M phosphate buffer, e g a 0.1 M phosphate buffer.

In an embodiment of the process according to the invention, the step (b) of incubating is carried out at room temperature or at lower temperatures. While the resulting crystal quality is better at 15-25°C, the yield is higher at lower temperatures, e g around 6°C. In practice, the employed temperature is a compromise between these two factors. The temperature may also be varied throughout the experiment. By way of example, the process can be started at a higher temperature, e g 15-25°C, for a period of time, e g 12 h, followed by a linear decrease in temperature down to 6°C over a period of time, e g 12 h. A suitable total incubation time is 12 h-96 h, preferably 24-48 h.

In an embodiment of the inventive process, the step (a) of preparing an aqueous solution of GH, or functional derivatives thereof, involves preparing a solution with an initial GH concentration above 0.1 mg/ml, such as in the range of from 0.1 to 20 mg/ml. Typically, the initial GH concentration is above 1 mg/ml, such as from 1 to 20 mg/ml, more preferably from 12 to 18 mg/ml. It is understood that if the weight or the solubility of any GH functional derivatives varies considerably from that of native GH, these values are easily altered accordingly. In a preferred embodiment of the process according to the invention, the GH is human GH, derived from natural or, preferably, recombinant sources.

In the process according to the invention, it has been found that the presence of cations in the aqueous solution of step (a) deteriorates the quality of the crystals, or even prevents their formation. In particu- lar, Zn²⁺ ions are especially effective in preventing crystal formation. This finding is in glaring contrast to the teachings of US 5,780,599, wherein the presence of Zn⁺ ions has a beneficial effect on GH crystal formation. Hence, in preferred embodiments of the process ac- cording to the invention, the concentration of divalent inorganic cations is lower than 0.3 mM, preferably lower than 0.1 mM, in the solution of step (a) . In a preferred embodiment, the solution of step (a) is void of divalent inorganic cations. The desire to exclude divalent inorganic cations may also be expressed as the molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, in the aqueous solution of step (a) . In a preferred embodiment, this molar ratio is less than 0.5, preferably less than 0.2. In a more preferred embodiment, the molar ratio is less than 0.1, preferably less than 0.003. As discussed above, it is particularly desirable to maintain a low concentration of zinc ions in the aqueous solution of step (a) . Accordingly, in preferred embodiments of the process according to the invention, the concentration of Zn²⁺ is lower than 0.002 mM in the solution of step (a) . In a preferred embodiment, the solution of step (a) is void of divalent zinc ions.

The desire to exclude Zn²⁺ may also be expressed as the molar ratio of Zn²⁺ to GH, or functional derivatives thereof, in the aqueous solution of step (a) . In a pre- ferred embodiment, this molar ratio is less than 0.003. The term "void of" in relation to zinc ions and other divalent inorganic cations, refers to the situation where no such ions have been intentionally added, i e that the concentrations of these ions are at or below the naturally occurring concentrations thereof in ordinary water.

The term "molar ratio" refers to the molar relationship between two species. In the present invention, it is understood that the molar ratio of divalent inorganic cations, such as Zn²⁺, to GH in the solution may be subject to change (increase) as GH crystals leave the liquid phase .

It is known that the availability of divalent inorganic cations in solution may be decreased through the use of chelating agents, such as EDTA and EGTA. These agents forms complexes with the divalent inorganic cations and provide a lowered effective concentration of the cations in the solution. Moreover, certain buffers, including phosphate and citrate buffers, possess chelat- ing properties. It is therefore contemplated that the effects of unwanted high concentrations of divalent inorganic cations in the aqueous solution may be diminished through the use of chelating agents in the present invention. Thus, the incorporation of divalent inorganic cations, including Zn²⁺, in combination with a chelating agent into the solution is anticipated. Nevertheless, it is preferable to omit these compounds and simply avoid or limit the use of divalent inorganic cations, including Zn²\

For the sake of clarity, it should be emphasized that the concentrations and molar ratios specified in the claims refer to the situation in the aqueous solution immediately prior to crystallization. In a batch crystallization, which is convenient for industrial manufacturing, the concentrations and molar ratios of the protein to be crystallized and any reagents, including the crystallizing agent, follow from the preparation of the solution.

However, in diffusion-based systems, the corresponding concentrations and ratios in the prepared solution containing the desired protein are subject to changes following equilibration with the reservoir solution. Nevertheless, the concentrations and ratios obtained are readily predictable, through the well-known fact that any volatile species will diffuse and come into equilibrium between the drop and the reservoir solution. Therefore, the final volume will be determined by the non-volatile species. In practice, this means that the concentrations and molar ratios in the aqueous solution immediately prior to crystallization are inherent in the set-up of the experiment, including the composition and starting volume of the drop, and the composition of the reservoir solution. Analogously, in dialysis-based systems, the corresponding concentrations and molar ratios in the aqueous solution immediately prior to crystallization are inherent in the set-up of the experiment. As the crystallization proceeds, the concentration of soluble GH will obviously decrease. The present invention shall not be considered to be limited to the above described method. According to another aspect, the invention provides a novel use of 2-propanol in an aqueous solution comprising growth hor- mone (GH) , or functional derivatives thereof, for manufacturing crystals of GH, or functional derivatives thereof, from said aqueous solution.

As mentioned previously, the characteristics and yield of the resulting crystals are further influenced by several parameters, including concentrations of

2-propanol and GH, or functional derivatives thereof, the presence and concentrations of buffers and divalent inorganic cations, particularly Zn²⁺, pH and temperature. In particular, it is preferred that the solution of step (a) is having a molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, of less than 0.5 and a molar ratio of zinc ions to GH, or functional derivatives thereof, of less than 0.2.

As an alternative, it is preferred that the concen- tration of divalent inorganic cations is lower than about 0.3 mM, and that the concentration of zinc ions is lower than about 0.002 mM in the solution of step (a) . It is particularly preferred that the solution is substantially void of divalent inorganic cations, particularly zinc ions.

A further embodiment of the invention provides crystals of GH, or functional derivatives thereof, manufactured by the process according to the invention. Without being limited thereto, the resulting crystals may be rod- shaped, but of varying crystal sizes and more detailed structures. The crystals may be useful as a medicament in the treatment of mammals, including humans. Optionally, the crystals may be useful for the manufacture of a medicament for treating a mammal, including man, in need of GH, or functional derivatives thereof. It is contemplated that the resulting crystals are useful for pharmaceutical purposes. Accordingly, an embodiment of the invention provides a pharmaceutical composition comprising a therapeutically effective amount of crystals of growth hormone (GH) , or functional derivatives thereof, according to the invention, and a suitable pharmaceutical carrier therefor. The composition may be suitable for any of several known administration forms, including topical, nasal, pulmonal, oral, rectal and par- enteral administration. Without being limited thereto, the composition may be a suspension for injection, a depot formulation or a dry formulation comprising crystals, which will have to be reconstituted before use.

Suitable pharmaceutical carriers for compositions of GH are well known to the skilled man in the field.

A further embodiment of the invention provides a method of treating a mammal, including man, in need of growth hormone (GH) , or functional derivatives thereof. The method comprises administration of a therapeutically effective amount of the crystals of GH, or functional derivatives thereof, to the mammal. The administration may be through any of the following routes: topical, nasal, pulmonal, oral, rectal and parenteral. In a preferred embodiment, the administration is performed parenterally.

In an embodiment of the invention, the process is useful for purification of GH from a solution thereof containing unwanted impurities.

As mentioned previously, in US 5 780 599 to Novo

Nordisk A/S, a method of producing growth hormone cation crystals is disclosed. The method comprises the steps: addition of divalent inorganic cations to a solution of GH at a pH between 5 and 8, growing of crystals at a tem- perature of 0°C-30°C, and isolation of the crystals. The obtained crystals always include a divalent inorganic cation, preferably Zn⁺. It is described that incubation of a solution of 0.27 mM hGH and 0.36 mM Zn²⁺ (Zn²⁺/hGH molar ratio of approximately 1.34) provides crystals of GH. It is further suggested that an inorganic cation²⁺/GH molar ratio of not less than 0.5, or a Zn²⁺/GH molar ratio of not less than 0.2, provides a suitable concentration of Zn²⁺ or divalent inorganic cations, respectively.

In the process according to the present invention, it has been found that when 2-propanol per se is employed as crystallizing agent, the presence of divalent inor- ganic cations in fact prevents the formation of crystals or deteriorates the quality of any crystals produced. In the following Example 6, it was found that as little as 0.002 M Zn²⁺ ( [Zn²⁺] / [hGH] ratio of approximately 0.003) prevented crystallization in the presence of 2-propanol. In the following Example 7, it was found that when

2-propanol is employed as crystallizing agent, the presence of the divalent inorganic cations Fe²⁺ (0.2-0.8 mM) , Mg²⁺ (4-10 mM) , and Ca²⁺ (1-3 mM) deteriorates the quality of the crystals formed, if any. It is therefore desirable to omit or limit the presence of these ions in the crystallization solutions according to the invention. The molar ratios were as follows: Fe²⁺/GH: 0.18-0.73, Mg²⁺/GH: 3.7-9.2, and Ca²⁺/GH: 0.92-2.8.

In a suggested embodiment of US 5 780 599, an or- ganic solvent is added together with the cation. While the experiments are concerned with the organic solvents acetone and ethanol, it is briefly proposed that 2-propanol is also a suitable organic solvent in conjunction with divalent inorganic cations. In the process according to the invention, it is experimentally shown that 2-propanol is a suitable crystallization agent per se, and that the presence of divalent inorganic cations, particularly zinc ions, deteriorates the quality of the resulting crystals of GH, or deriva- tives thereof.

Accordingly, US 5 780 599 does not teach the use of 2-propanol as a suitable crystallization agent per se . In contrast, US 5 780 599 teaches use of high concentrations of zinc ions as crystallizing agent, optionally in conjunction with certain organic solvents.

The present invention will in the following be further illustrated by examples. The examples should not be interpreted as limiting the scope of the invention.

EXAMPLES In the following, Examples 1-7 refer to crystal screening experiments, Examples 8-15 refer to stock crystal batch experiments, and Examples 16-23 refer to GH solubility experiments.

EXAMPLE 1 Preparation of hGH

All the hGH material which was used in the crystal screening was dialyzed against water. The resulting sample was filtered through a 0.45 μm cellulose acetate membrane to remove possible solid impurities and to prevent microbial contamination. The resulting hGH solution was diluted with water to yield a solution^' with desired protein concentration.

EXAMPLE 2 Crystal screening of 2-propanol concentration and pH

2-propanol was screened, using the hanging drop vapor diffusion technique, at concentrations in the range of from 5% to 30 % (v/v) and pH values in the range of from 5.3 to 7.8. Briefly, 5 μl sample containing 33 mg/ml GH and 5 μl reagent solution containing 5, 10, 20 or 30% (v/v) 2-propanol in 20 mM Na-K-phosphate, pH 5.3, 5.6, 6.0, 6.4, 7.0, or 7.8, were pipetted and mixed on a circular siliconized cover slide. The slide was placed on the top of a tissue culture box with 24 wells and sealed with grease . The chamber contained 1 ml of the same reagent solution (called a reservoir solution) . Following equili- bration, the reagent concentration in the drop is nearly the same as that in the chamber. Thus, the final volume of the drop is approximately the same as the volume of reagent solution which was initially pipetted (5 μl) , and it contains approximately 20 mM Na/K phosphate, 33 mg/ml GH, and from 5% to 30% (v/v) 2-propanol.

The drops were evaluated frequently by microscopy. Some interesting objects were photographed using an Olympus digital camera on a microscope. The results are presented in Table 1.

TABLE 1

Following 4 days of incubation, amorphous precipitate or liquid phase separation appeared in the drops with pH 5.3 and pH 5.6. After 11 days, crystals were observed in addition to amorphous precipitate in the drops with 5-20% 2-propanol. The crystals were mostly very small needles. In 20% concentration at pH 5.3, there were also rods, and at pH 5.6, very large rods in addition to small needles. The drop with 20% 2-propanol and pH 6.0 contained few crystals but no amorphous precipitate after 11 days.

EXAMPLE 3 Crystal screening of 2-propanol concentration and pH

2-propanol was further screened, using the hanging drop vapor diffusion technique, at concentrations in the range of from 14% to 20 % (v/v) and pH values in the range of from 5.6 to 7.0.

Briefly, 5 μl sample containing 17 mg/ml GH and 5 μl reagent solution containing 14, 16, 18 or 20% (v/v) 2-propanol in 0.1 M Na/K phosphate, pH 5.6, 6.0, or 7.0, were mixed and treated as described in Example 2. The results are presented in Table 2.

TABLE 2

Following 12 days of incubation, short and thick rod-shaped crystals were observed in the drops with 18% 2-propanol at pH 5.6. After 19 days of incubation, short and thick rod-shaped crystals had appeared in the drops with 16% 2-propanol, pH 5.6.

EXAMPLE 4 Crystal screening of buffer strength Phosphate was screened in combination with 2-propanol. Phosphate was screened at four concentrations from 0.4 M to 0.7 M and three pH values from 7.0 to 8.2.

The same conditions were screened with 15% and 20% (v/v)

2-propanol. The final protein concentration was 17 mg/ml.

Briefly, 5 μl sample containing 17 mg/ml GH and 5 μl reagent solution containing 15 or 20% (v/v) 2-propanol in

0.4, 0.5, 0.6 or 0.7 M Na-K-phosphate, pH 7.0, 7.8 or

8.2, were mixed and treated as described in Example 2.

The results are presented in Table 3. TABLE 3

20% 2-propanol

Microscopy observations C = crystals

A = amorphous precipitate L = liquid phase separation, spherical droplets G = gel, glassy solid irregular particles

N = no phase separations, clear solution

X = experiment failed, discontinued, dried, microbial contamination, etc.

In two days, amorphous precipitate appeared in the drops with 0.7 M phosphate, 20% 2-propanol at pH 7.8 and pH 8.2. After five days of incubation, needle-shaped crystals were observed in the drops with 15% 2-propanol and pH 7.8 or pH 8.2. The drops with 0.6 M phosphate, 20% 2-propanol at pH 7.8 and pH 8.2 contained liquid phase separation and very small needles. After 23 days of incubation, almost all the drops contained crystals. The drops with highest phosphate concentration contained liquid phase separation and very small needles or amorphous precipitate.

EXAMPLE 5 Crystal screening of buffer strength Phosphate was screened in combination with

2-propanol. Phosphate was screened at four concentrations from 0.5 M to 0.8 M and three pH values from 7.0 to 8.2. The same conditions were screened with 10% (v/v) 2-propanol and in the absence of 2-propanol. The final protein concentration was 17 mg/ml.

Briefly, 5 μl sample containing 17 mg/ml GH and 5 μl reagent solution containing 0 or 10% (v/v) 2-propanol in 0.5, 0.6, 0.7 or 0.8 M Na-K-phosphate, pH 7.0, 7.8 or 8.2, were mixed and treated as described in Example 2. The results are presented in Table 4. TABLE 4

X = experiment failed, discontinued, dried, microbial contamination, etc

Needle-shaped crystals were obtained in most of the drops with 2-propanol. In the drops which did not contain 2-propanol, mostly amorphous precipitate and at best very small needles were formed. At pH 8.2, the drops with 2-propanol also contained a lot of amorphous precipitate.

EXAMPLE 6 Crystal screening of ZnCl₂

ZnCl₂ was screened in combination with 2-propanol. ZnCl₂ was screened at four concentrations from 0.002 mM to 0.20 mM and three pH values from 5.6 to 7.0. The same conditions were screened with 10% and 15% (v/v) 2-propanol. 20 mM bis-Tris-HCl buffer was used to control pH.

Briefly, 5 μl sample containing 17 mg/ml GH and 5 μl reagent solution containing 0.002, 0.01, 0.05 or 0.2 mM ZnCl₂ and 10 or 15% (v/v) 2-propanol in 0.1 M Na-K- phosphate, pH 5.6, 6.0 or 7.0, were mixed and treated as described in Example 2. The results are shown in Table 5.

TABLE 5

Following 19 days of incubation, most of the drops contained amorphous precipitate or gel. No crystals were obtained.

Thus, the presence of Zn²⁺ ions, at a concentration in the range of 0.002-0.2 mM, prevents the formation of crystals at the conditions tested. It is therefore desirable to omit or limit the presence of these ions in the crystallization solutions according to the invention. Notably, the molar ratios Zn²⁺/GH were in the range of 0.003-0.26.

EXAMPLE 7 Crystal screening of divalent metal ions

Three divalent metal ions, Fe²⁺, Mg²⁺, and Ca²⁺, were screened at pH values 5.6 and 6.0. The samples were screened in the absence of 2-propanol or in the presence of 18% (v/v) 2-propanol.

Briefly, 5 μl sample containing 24 mg/ml GH and 5 μl reagent solution containing 0.2 or 0.8 mM Fe(II)S0₄, 4.0 or 10 mM MgCl₂, or 1.0 or 3.0 mM CaCl₂, and 10 or 15% (v/v) 2-propanol in 10 mM bis-Tris-HCl, pH 5.6 or 6.0, were mixed and treated as described in Example 2. The results are shown in Table 6.

TABLE 6

The divalent metal ions used alone as crystallizing agents did not produce crystals, only liquid phase separation, gel or amorphous precipitate was observed. To- gether with 2-propanol, rod- or needle-shaped crystals were obtained, but there was also amorphous precipitate present in most of the drops . There was no change in the results after up to 41 days of incubation.

Thus, the presence of the divalent inorganic cations Fe²⁺ (0.2-0.8 mM) , Mg²⁺ (4-10 mM) , and Ca²⁺ (1-3 mM) deteriorates the quality of the crystals formed, if any. It is therefore desirable to omit or limit the presence of these ions in the crystallization solutions according to the invention. Notably, the molar ratios were as follows: Fe²⁺/GH: 0.18-0.73, Mg²⁺/GH : 3.7-9.2, and Ca²⁺/GH: 0.92- 2.8.

EXAMPLE 8 Preparation of a crystal minibatch with 2-propanol The 2-propanol system was tested in batches of a few milliliters. Two 2-propanol concentrations, 16 and 18% (v/v), and two pH values, pH 5.6 and pH 5.8, were used. The protein solution and stock crystallization medium were mixed in equal proportions in order to achieve the final concentration.

In general, small scale batch crystallizations were performed in syringes. Crystallizations were performed by mixing the reagents in a small beaker with a magnetic stirrer. The mixture was then drawn into a syringe, air bubbles were removed from the mixture, and the syringe was sealed with a cap. The syringe was placed on a vertical rotator (16 rpm) . The idea of this gentle method was to minimize the air/liquid interface, to ensure complete mixing and to prevent the formation of sediment. For crystallizations in 16% 2-propanol, the stock crystallization medium contained the following reagents: 32% (v/v) 2-propanol and 0.2 M phosphate, pH 5.6 or 5.8. For crystallizations in 18% 2-propanol, the stock crystallization medium contained the following reagents: 36% (v/v) 2-propanol and 0.2 M phosphate, pH 5.6 or 5.8.

1.5 ml of hGH, dialyzed in water (24.1 mg/ml), was pipetted into a small beaker with a magnetic stirrer. 1.5 ml of crystallization stock reagent was added dropwise under continuous stirring. Following addition of the medium, the solution was drawn into a syringe and placed on a vertical rotator at room temperature.

The soluble protein content was determined the following day. The samples were observed and any crystals formed were studied in more detail under a microscope. The results are displayed in Table 7.

TABLE 7

The sample with 16 % (v/v) 2-propanol and pH 5.6 started to crystallize within a few hours. The crystals were thick rods with sharp pointed ends. All the samples contained crystals the following day. Most of the crystals in the samples with 16% 2-propanol were thick rods. The sample with 16% 2-propanol and pH 5.8 contained also thin rods and some gel lumps. The crystals in the samples with 18% 2-propanol were mostly thin rods or needles. Some thick rods with pointed ends were also observed. The crystal yields varied between 71% and 94%. The best yield was obtained in the sample with 18% 2-propanol at pH 5.6. The best quality of the crystals was achieved in the sample with 16% 2-propanol at pH 5.6. 2-propanol produced rod-shaped crystals with sharp pointed ends, and the thickness of the crystals seems to be very dependent on the crystallization conditions. The results show that batch crystallizations can be made in 2-propanol with good yield.

EXAMPLE 9 Preparation of a stock crystal batch in 2-propanol

100 ml of hGH solution (23.7 mg/ml) was poured into a 250 ml round-bottomed reaction flask. A tube magnetic mixer (PAM Solutions Ltd Oy) was activated. 100 ml of stock crystallization medium (36% 2-propanol, 0.2 M phosphate, pH 5.6) was added in -20 ml portions. The solution started to precipitate after 40 ml of reagent had been added. Following addition of the remaining stock crystallization medium, the precipitate was observed with a microscope. The precipitate consisted of very small crystals or gel lumps.

The following day, the batch contained rod-shaped crystals and crystal clusters. Only a few gel lumps were observed. The final soluble protein concentration was 1.1 mg/ml, and the crystal yield was 91%. The batch was concentrated by sedimenting the crystals and removing -150 ml of the clear mother liquor. The total protein concen- tration of the concentrated crystal slurry was 40.3 mg/ml .

EXAMPLE 10 Preparation of a stock crystal batch in 2-propanol A stock crystal batch was prepared in the way described in Example 9, with the exceptions that the hGH starting material had a concentration of 26.3 mg/ml, and the addition of the stock crystallization medium was performed with a peristaltic pump during 1.5 hours. The batch started to precipitate when approximately one third of the stock crystallization medium was added. After a few hours, the batch was observed with a microscope, and was found to contain gel lumps.

The following day, most of the gel lumps in the batch had turned into thin rod-shaped crystals. The gel lumps on the object glass had turned into very large rods during the night . The final soluble protein concentration was 0.74 mg/ml, and the crystal yield was 94%. The batch was concentrated by sedimenting the crystals and removing -100 ml of the clear mother liquor. The total protein concentration of the concentrated crystal slurry was 23.3 mg/ml .

EXAMPLE 11 Preparation of a stock crystal batch in 2-propanol A stock crystal batch was prepared in the way described in Example 10, with the exceptions that the 2-propanol concentration was raised from 18% to 20% in order to increase the crystal yield.

80 ml of hGH raw material (31.2 mg/ml) was poured into a 250 ml round-bottomed reaction flask. 80 ml of stock crystallization medium (40% 2-propanol, 0.2 M phosphate, pH 5.6) was added with a peristaltic pump for a period of a few hours. The solution started to precipitate after one third of the reagent volume was added. A few hours after the reagent had been added, the precipitate was observed with a microscope, and was found to contain thin clustered crystals and some larger rod- shaped crystals. The following day, the amount of crystals had increased. The final soluble protein concentra- tion was 1.3 mg/ml, and the crystal yield was 92%. After two days, the crystal slurry was concentrated by sedimenting and removing supernatant. The total protein concentration of the concentrated crystal slurry was 55.6 mg/ml . EXAMPLE 12 Batch crystallization in 2-propanol at low temperature

A batch crystallization in 2-propanol was prepared at +2° C. 30 ml of hGH material was poured into a reac- tion flask, and an equal amount of stock crystallization medium (36% 2-propanol, 0.2 M phosphate, pH 5.6) was added by pouring slowly. An amorphous or gel -like precipitate was formed during the addition.

The precipitate in the batch did not turn into crys- tals during the following night. The batch contained large gel particles instead of crystals. The soluble protein concentration was 0.54 mg/ml. The pH of the batch was measured to be 6.2.

EXAMPLE 13 Batch crystallization in 2-propanol at low temperature

A batch crystallization in 2-propanol was prepared at +8° C. 20 ml of hGH material was poured into the reaction flask, and an equal amount of stock crystallization medium (36% 2-propanol, 0.2 M phosphate, pH 5.6) was added by pouring slowly. An amorphous or gel -like precipitate was formed during the addition.

Following the first night, the precipitate was still a gel. The soluble protein concentration was 1.8 mg/ml. The batch was seeded with 200 μl of crystal slurry from the batch in Example 11. During the second night, the precipitate turned into rod-shaped crystals. The pH of the batch was measured to be 6.2. The soluble protein concentration was 1.7 mg/ml .

EXAMPLE 14 Batch crystallization in 2-propanol at low temperature

A batch crystallization in 2-propanol was prepared at +15° C. 20 ml of hGH material was poured into the re- action flask, and an equal amount of stock crystallization medium (36% 2-propanol, 0.2 M phosphate, pH 5.6) was added by pouring slowly. An amorphous or gel-like precipitate was formed during the addition.

Following the first night, the precipitate was still a gel. The soluble protein concentration was 1.5 mg/ml. The batch was seeded with 200 μl of crystal slurry from the batch in Example 11. During the second night, the precipitate turned into thin needle-shaped crystals. The pH of the batch was measured to be 6.3. The soluble protein concentration was 0.5 mg/ml.

EXAMPLE 15 Incubation of crystals at reduced temperatures

In Example 14, the batch prepared in 2-propanol at +15°C yielded crystals. To see whether these crystals would change their appearance at lower temperatures, the temperature was decreased gradually, and the crystals were observed by microscopy. The crystals were incubated for at least one night at each temperature.

During the period of temperature reduction, the crystals did not change their appearance much. The soluble protein concentration remained very low at all the tested temperatures. The results are shown in Table 8.

Table 8

In 2-propanol, crystallization was successful at

+8°C and higher temperatures. The storage of crystals at refrigerator temperatures should be possible. According to phase diagrams (Example 23, Fig 5-7), it should be possible to perform also the crystallization even at lower temperatures. This may require simple adjusting of other conditions, like 2-propanol concentration, pH, etc.

EXAMPLE 16 Preparation of crystal suspensions for solubility experiments: the washing method

An aliquot of a stock crystal slurry was washed for each experimental condition. Experiments were made in 1.5 ml Eppendorf tubes. 800 μl of crystal slurry and 500 μl of reagent solution were mixed in the tube. The crystals were centrifuged (8832 g, 1 min) , and the supernatant was discarded. 750 μl of reagent solution was added, and the crystals were suspended with a plastic stick. Centrifug- ing and discarding of the supernatant was repeated. Finally, 1500 μl of reagent solution was added, and the crystals were suspended. The tubes were kept in a thermo- stated chamber at +20° C. The samples were incubated for at least three days to achieve equilibrium.

EXAMPLE 17 Preparation of crystal suspensions for solubility experiments: the dialysis method

Crystals were dialyzed into the experimental conditions. 10 ml of reagent solution was pipetted into a glass vial. 1 ml of crystal suspension was pipetted into a dialysis tube (MWCO 6000-8000, 0.32 ml/cm). One end of the tube was sealed by knotting, and the other end was sealed by placing it between the vial and the rubber stopper, which was used to close the vial. The vials were shaken gently on an orbital shaker in a thermostated chamber at +20° C. The dialysis solution was replaced with fresh reagent solution after four hours. The samples were incubated for at least three days to achieve solubility equilibrium. For temperature experiments, the crystal slurry, which was dialyzed to the desired chemical conditions, was divided into small glass vials and incubated in a thermostated chamber at various temperatures for at least two days .

EXAMPLE 18 Preparation of crystal suspensions for solubility experiments: the dilution method

A concentrated stock crystal slurry was diluted with suitable reagents to achieve the reagent conditions in the experimental points. The samples were incubated for at least three days to achieve equilibrium.

EXAMPLE 19 The effect of 2-propanol concentration on the solubility of hGH crystals

Using either the washing method of Example 16 or the dialysis method of Example 17, a series of solubility experiments were performed at +20°C in a reagent solution containing 0.1 M phosphate, 5-30% (v/v) 2-propanol, pH 5.3-8.0. The GH crystals were allowed to achieve solubility equilibrium in the various reagent solutions, and the resulting soluble hGH concentrations were determined. All the protein assays were performed by measuring the ab- sorbance value at wavelength 280 nm. The absorbance values were converted to dry matter by dividing with the factor 0,73.

The results are shown in Table 9 and illustrated graphically in Fig 1 and 3.

Table 9

¹ The methods to prepare the experimental points are described in Examples 16 and 17. W means washing method and D means dialysis method.

² Points in which the crystals had dissolved or turned into gel granules were excluded from the figures.

The combined effect of 2-propanol concentration and pH was complex. A solubility minimum was found around 20% (v/v) 2-propanol at all pH values. Increasing 2-propanol concentration decreased the solubility of hGH crystals up to a 2-propanol concentration of 20%. At higher 2- propanol concentrations than 20%, the solubility increased again. At a 2-propanol concentration of 30%, the soluble protein concentrations were lower than at 25% 2-propanol, but the crystals had turned to gel granules and thus, the solubilities are not comparable to the solubilities of the crystals. These experimental points were excluded from the figures.

Typically, the solubility of protein decreases when the concentration of crystallization agent increases.

However, in 2-propanol, a solubility minimum could be observed around 20 %.

EJXAMPLE 20 The effect of phosphate concentration on the solubility of hGH crystals in 2-propanol

Using the dialysis method of Example 17, a series of solubility experiments were performed at +20°C in a reagent solution containing 0.4-0.7 M phosphate, 10% or 15% (v/v) 2-propanol, pH 5.3 or 7.8. The GH crystals were al- lowed to achieve solubility equilibrium in the various reagent solutions, and the resulting soluble hGH concentrations were determined. The results are shown in Table 10 and illustrated graphically in Fig 2. Table 10

The solubility of hGH crystals decreased with increasing phosphate concentration in 2-propanol. At pH 5.3, the change between the solubility in 0.4 M and 0.7 M phosphate was small. At pH 7.8 in 10% 2-propanol, the solubility decreased significantly. The crystals in pH 5.3 were large and well-shaped when observed after the experiment. The crystals in pH 7.8 had changed; they were a lot smaller and there was some gel which glued some of the crystals into clusters. Gel formation may have had some influence on the shape of the solubility curve.

EXAMPLE 21 The effect of pH on the solubility of hGH crystals in 2-propanol

The data obtained in Example 19 may also be illustrated graphically as the effect of pH (5.3-8.0) on the solubility of hGH crystals in various 2-propanol concentrations (5-25%) . This is shown in Fig 3 (c f Table 9 and Fig 1) .

The effect of pH on the solubility of hGH crystals was rather unusual. The solubility increased sharply with increasing pH value until it reached a maximum at pH 6.5. The maximum solubility range was around pH 6.5 in all the 2-propanol concentrations. At pH values 7.0 and 8.0, the solubility was much lower again, but the solubility was still too high to be of immediate interest. The crystals did not change their form at high pH values .

The general effect of pH was that when pH was increasing, the solubility was also increasing at all 2-propanol concentrations. At low pH values, the range of low solubility became broader, and the solubility was low even at a 2-propanol concentration of 10%.

EXAMPLE 22 The effect of pH on the solubility of hGH crystals in phosphate combined with 2-propanol Using the dialysis method of Example 17, a series of solubility experiments were performed at +20°C in a reagent solution containing 0.4 or 0.7 M phosphate, 15% (v/v) 2-propanol, pH 5.3-7.0. The GH crystals were allowed to achieve solubility equilibrium in the various reagent solutions, and the resulting soluble hGH concentrations were determined. The results are shown in Table 11 and illustrated graphically in Fig 4.

Table 11

The solubility of hGH crystals in 0.4 M and 0.7 M phosphate in 15% 2-propanol increased with increasing pH value, until it reached a maximum at pH 6.2 (Fig 4) . Af- ter the maximum, the solubility decreased again. In 0.1 M phosphate, the maximum was at a somewhat higher pH value, pH 6.5. This kind of protein solubility curve is uncommon.

EXAMPLE 23 The effect of temperature on the solubility of hGH crystals in 2-propanol

Using the dialysis method of Example 17, a series of solubility experiments were performed at various temperatures in the range of 2-30° C in a reagent solution con- taining 0.1 M phosphate, 10% or 20% (v/v) 2-propanol, pH 5.3 or 5.7. The GH crystals were allowed to achieve solubility equilibrium in the various reagent solutions, and the resulting soluble hGH concentrations were determined. The results are shown in Table 12 and illustrated graphically in Fig 5.

The solubility of hGH crystals in 2-propanol increased with increasing temperature. At pH 5.7, the temperature effect was stronger than at pH 5.3

EXAMPLE 24 The effect of temperature on the solubility of hGH crystals in phosphate combined with 2-propanol

Using the dialysis method of Example 17, a series of solubility experiments were performed at various temperatures in the range of 2-30° C in a reagent solution con- taining 0.4 or 0.8 M phosphate, 10% or 20% (v/v)

2-propanol, pH 5.7. The GH crystals were allowed to achieve solubility equilibrium in the various reagent solutions, and the resulting soluble hGH concentrations were determined. The results are shown in Table 13 and illustrated graphically in Fig 6 (10% 2-propanol) and Fig 7 (20% 2-propanol) .

The solubility of hGH crystals in phosphate combined with 2-propanol increased with increasing temperature (Fig 6 and 7) . The solubility differences between various phosphate concentrations are greater in 10% 2-propanol than in 20% 2-propanol. Crystals turned into gel at high- est temperatures in 20% 2-propanol at 0.4 M and 0.8 M phosphate concentration. The solubility of the gel is not comparable to the solubility of the crystals and thus, these results were excluded from the figures.

Claims

1. A process for manufacturing crystals of growth hormone (GH) , or functional derivatives thereof, compris- ing the following steps:

(a) preparing an aqueous solution of GH, or functional derivatives thereof, comprising 2-propanol, such that the molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, is less than 0.5, and the mo- lar ratio of zinc ions to GH, or functional derivatives thereof, is less than 0.003 in said solution,

(b) incubating the solution prepared in step (a) to crystallize said GH, or functional derivatives thereof, and

(c) isolating the crystals formed in step (b) . 2. A process according to claim 1, wherein said

2-propanol is present in a concentration of at most 25% v/v.

3. A process according to claim 2, wherein said 2-propanol is present in a concentration in the range of 5-25% v/v.

4. A process according to claim 3, wherein said 2-propanol is present in a concentration in the range of 16-20% v/v.

5. A process according to claim 4, wherein said 2-propanol is present in a concentration in the range of

18-20% v/v.

6. A process according to any one of claims 1-5, wherein the solution prepared in step (a) also comprises a buffer selected from sodium/potassium phosphate, citric acid, a combination of sodium/potassium phosphate and citric acid, and BisTris-HCl.

7. A process according to claim 6, wherein said buffer is sodium/potassium phosphate.

8. A process according to claim 6 or 7 , wherein said buffer is present in a concentration of more than 10 mM.

9. A process according to claim 8, wherein said buffer is present in a concentration range of 20 mM-0.8 M.

10. A process according to claim 9, wherein said buffer is present in a concentration range of 20 mM-0.1 M.

11. A process according to any one of claims 1-10, wherein the solution prepared in step (a) is adjusted to a pH of less than about 6.2.

12. A process according to claim 11, wherein the solution prepared in step (a) is adjusted to a pH in the range of 5.5-6.2.

13. A process according to claim 12, wherein the solution prepared in step (a) is adjusted to a pH in the range of 5.5-5.7.

14. A process according to any one of claims 1-10, wherein the solution prepared in step (a) is adjusted to a pH of more than about 7.0.

15. A process according to any one of claims 1-14, wherein said GH, or functional derivatives thereof, is present in the solution prepared in step (a) in a concentration above 0.1 mg/ml.

16. A process according to claim 15, wherein said concentration of GH, or functional derivatives thereof, is above 1 mg/ml.

17. A process according to claim 16, wherein said concentration of GH, or functional derivatives thereof, is in the range of 1-20 mg/ml.

18. A process according to claim 17, wherein said concentration of GH, or functional derivatives thereof, is in the range of 12-18 mg/ml.

19. A process according to any one of claims 1-18, wherein said GH is human GH.

20. A process according to any one of claims 1-19, wherein said molar ratio of divalent inorganic cations to

GH, or functional derivatives thereof, is less than 0.2.

21. A process according to claim 20, wherein said molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, is less than 0.1.

22. A process according to claim 21, wherein said molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, is less than 0.003.

23. A process according to any one of claims 1-22, wherein the concentration of divalent inorganic cations is lower than about 0.3 mM in said solution of step (a) .

24. A process according to claim 23, wherein the concentration of divalent inorganic cations is lower than about 0.1 mM in said solution of step (a) .

25. A process according to claim 24, wherein said solution of step (a) is substantially void of divalent inorganic cations.

26. A process according to any one of claims 1-25, wherein the concentration of zinc ions is lower than about 0.002 mM in said solution of step (a) .

27. A process according to claim 26, wherein said solution of step (a) is substantially void of zinc ions.

28. Use of 2-propanol as a crystallizing agent for manufacturing crystals of GH, or functional derivatives thereof .

29. Use of 2-propanol in an aqueous solution com- prising growth hormone (GH) , or functional derivatives thereof, said solution having a molar ratio of divalent inorganic cations to GH, or functional derivatives thereof, of less than 0.5 and a molar ratio of zinc ions to GH, or functional derivatives thereof, of less than 0.003, for manufacturing crystals of GH, or functional derivatives thereof, from said aqueous solution.

30. Crystals of GH, or functional derivatives thereof, obtainable by the process according to any one of claims 1-27.

31. A pharmaceutical composition comprising a therapeutically effective amount of crystals of growth hormone (GH) , or functional derivatives thereof, according to claim 30, and a suitable pharmaceutical carrier therefor.

32. A pharmaceutical composition according to claim 31, wherein said composition is a suspension for injec- tion.

33. A pharmaceutical composition according to claim 31, wherein said composition is a depot formulation.

34. A pharmaceutical composition according to claim 31, wherein said composition is a dry formulation.

35. Crystals of growth hormone (GH) , or functional derivatives thereof, according to claim 30, for use as a medicament .

36. Use of crystals of growth hormone (GH) , or functional _ derivatives thereof, according to claim 30, for the manufacture of a medicament for treating a mammal, including man, in need of GH, or functional derivatives thereof .

37. A method of treating a mammal, including man, in need of growth hormone (GH) , or functional derivatives thereof, comprising administering to said mammal, in need of such a treatment, a therapeutically effective amount of crystals of GH, or functional derivatives thereof, according to claim 30.