Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/61—Growth hormones [GH] (Somatotropin)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/27—Growth hormone [GH] (Somatotropin)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/10—Drugs for disorders of the endocrine system of the posterior pituitary hormones, e.g. oxytocin, ADH

Definitions

• 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.
• GH growth hormone
• 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.
• 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.
• 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.
• hGH recombinant 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 ⁇ -octyl glucoside.
• 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 . ..
• a method of producing chemically stable and biologically active growth hormone cation crystals 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 Zn 2+ .
• 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.
• 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.
• GH growth hormone
• 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) .
• said 2-propanol is present in a concentration of at most 25% v/v.
• 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.
• 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.
• said buffer is sodium/potassium phos- phate.
• 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.
• the solution prepared in step (a) is adjusted to a pH of less than about 6.2.
• 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.
• 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.
• 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 .
• 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.
• said GH is human GH.
• 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) .
• 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.
• 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) .
• said solution of step (a) is substantially void of divalent inorganic cations.
• the concentration of zinc ions is lower than about 0.002 mM in said solution of step (a) .
• said solution of step (a) is sub- stantially void of zinc ions.
• the present invention provides a novel use of 2-propanol as a crystallizing agent for manufacturing crystals of GH, or functional derivatives thereof.
• 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.
• 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.
• the present invention provides a pharmaceutical composition
• 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.
• said composition is a suspension for injection.
• said composition is a depot formulation.
• 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.
• 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.
• GH growth hor- mone
• 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.
• 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.
• 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) .
• 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.
• 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.
• 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 .
• 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.
• 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.
• 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.
• the protein 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 .
• a protein sample drop typically 5-30 ⁇ l
• 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.
• 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 .
• a small volume, such as 1-30 ⁇ l , of protein solution is mixed with a small volume, such as 1-30 ⁇ l, 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.
• 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 :
• step (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.
• 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.
• 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) .
• 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.
• crystals of GH are obtainable.
• 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%.
• 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.
• 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.
• 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.
• the aqueous solution of GH, or functional derivatives thereof is prepared such that the resulting pH is adjusted to less than about 6.2.
• 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.
• 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.
• 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.
• 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.
• the employed temperature is a compromise between these two factors.
• the temperature may also be varied throughout the experiment.
• 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.
• 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.
• 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.
• the GH is human GH, derived from natural or, preferably, recombinant sources.
• the concentration of divalent inorganic cations is lower than 0.3 mM, preferably lower than 0.1 mM, in the solution of step (a) .
• 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) .
• this molar ratio is less than 0.5, preferably less than 0.2.
• the molar ratio is less than 0.1, preferably less than 0.003.
• the concentration of Zn 2+ is lower than 0.002 mM in the solution of step (a) .
• the solution of step (a) is void of divalent zinc ions.
• the desire to exclude Zn 2+ may also be expressed as the molar ratio of Zn 2+ to GH, or functional derivatives thereof, in the aqueous solution of step (a) .
• this molar ratio is less than 0.003.
• 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.
• molar ratio refers to the molar relationship between two species.
• divalent inorganic cations such as Zn 2+
• GH in the solution may be subject to change (increase) as GH crystals leave the liquid phase .
• divalent inorganic cations 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.
• 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.
• the incorporation of divalent inorganic cations, including Zn 2+ 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 Zn 2 ⁇
• concentrations and molar ratios specified in the claims refer to the situation in the aqueous solution immediately prior to crystallization.
• concentrations and molar ratios of the protein to be crystallized and any reagents, including the crystallizing agent follow from the preparation of the solution.
• 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.
• 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.
• GH growth hor- mone
• the characteristics and yield of the resulting crystals are further influenced by several parameters, including concentrations 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.
• 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.
• 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.
• 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.
• 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.
• GH growth hormone
• 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.
• the process is useful for purification of GH from a solution thereof containing unwanted impurities.
• a method of producing growth hormone cation crystals 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 Zn 2+ (Zn 2+ /hGH molar ratio of approximately 1.34) provides crystals of GH.
• an inorganic cation 2+ /GH molar ratio of not less than 0.5, or a Zn 2+ /GH molar ratio of not less than 0.2 provides a suitable concentration of Zn 2+ or divalent inorganic cations, respectively.
• 2-propanol is employed as crystallizing agent, the presence of the divalent inorganic cations Fe 2+ (0.2-0.8 mM) , Mg 2+ (4-10 mM) , and Ca 2+ (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: Fe 2+ /GH: 0.18-0.73, Mg 2+ /GH: 3.7-9.2, and Ca 2+ /GH: 0.92-2.8.
• US 5 780 599 does not teach the use of 2-propanol as a suitable crystallization agent per se .
• US 5 780 599 teaches use of high concentrations of zinc ions as crystallizing agent, optionally in conjunction with certain organic solvents.
• Examples 1-7 refer to crystal screening experiments
• Examples 8-15 refer to stock crystal batch experiments
• Examples 16-23 refer to GH solubility experiments.
• hGH material which was used in the crystal screening was dialyzed against water.
• the resulting sample was filtered through a 0.45 ⁇ m 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.
• 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 ⁇ l sample containing 33 mg/ml GH and 5 ⁇ l 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) .
• a reservoir solution 1 ml of the same reagent solution
• the reagent concentration in the drop is nearly the same as that in the chamber.
• the final volume of the drop is approximately the same as the volume of reagent solution which was initially pipetted (5 ⁇ l) , and it contains approximately 20 mM Na/K phosphate, 33 mg/ml GH, and from 5% to 30% (v/v) 2-propanol.
• 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.
• A amorphous precipitate
• L liquid phase separation
• G gel, glassy solid irregular particles
• 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.
• ZnCl 2 was screened in combination with 2-propanol.
• ZnCl 2 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.
• the presence of Zn 2+ 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.
• the molar ratios Zn 2+ /GH were in the range of 0.003-0.26.
• the divalent metal ions used alone as crystallizing agents did not produce crystals, only liquid phase separation, gel or amorphous precipitate was observed.
• the presence of the divalent inorganic cations Fe 2+ (0.2-0.8 mM) , Mg 2+ (4-10 mM) , and Ca 2+ (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: Fe 2+ /GH: 0.18-0.73, Mg 2+ /GH : 3.7-9.2, and Ca 2+ /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.
• 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.
• 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.
• 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
• 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.
• 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.
• 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.
• 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.
• the precipitate was still a gel.
• the soluble protein concentration was 1.8 mg/ml.
• the batch was seeded with 200 ⁇ l of crystal slurry from the batch in Example 11.
• 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 .
• 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.
• the precipitate was still a gel.
• the soluble protein concentration was 1.5 mg/ml.
• the batch was seeded with 200 ⁇ l of crystal slurry from the batch in Example 11.
• 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 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.
• 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 .
• 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 16 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.
• solubility of protein decreases when the concentration of crystallization agent increases.
• the solubility of hGH crystals decreased with increasing phosphate concentration in 2-propanol.
• pH 5.3 the change between the solubility in 0.4 M and 0.7 M phosphate was small.
• 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 19 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) .
• 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.
• Example 17 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.

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