GB2371227A

GB2371227A - Crystallisation - resistant aqueous growth hormone formulations

Info

Publication number: GB2371227A
Application number: GB0100658A
Authority: GB
Inventors: Bernhard Siebold; John Stevens
Original assignee: Grandis Biotech GmbH
Current assignee: Grandis Biotech GmbH
Priority date: 2001-01-10
Filing date: 2001-01-10
Publication date: 2002-07-24
Also published as: GB0100658D0; WO2002067989A1

Abstract

The invention relates to aqueous growth hormone formulations comprising growth hormone and either citrate buffer at pH 5.6 or greater, or a non-citrate buffer at pH 6.0 or greater, which are substantially free of crystallisation on storage. Examples of such formulations use recombinant human growth hormone. Buffers other than citrate used include phosphate buffer, ammonium acetate and glycine. The formulations may in addition include agents for isotonicity, such as mannitol, propylene glycol, sodium chloride or ammonium chloride, non-ionic surfactants, such as Pluronic F-68 (RTM), and preservatives such as phenol or benzyl alcohol. The formulations are stable and exhibit little or no crystallisation when stored at temperatures above refrigeration temperatures, and may be used for the treatment of hypopituitary dwarfism.

Description

CRYSTALLISATION-RESISTANT AQUEOUS GROWTH HORMONE FORMULATIONS The present invention relates to liquid formulations of growth hormone (GH) which are storage stable, show reduced or no crystallisation on storage and are suitable for administration to the human or animal body. More particularly, the invention relates to liquid formulations of human growth hormone (hGH) which are stable and exhibit minimal or no crystallisation when stored at least for a time at temperatures above refrigeration temperatures.

Native hGH is a single polypeptide chain protein consisting of 191 amino acids. The protein is internally cross-linked by two disulphide bridges and in monomeric form exhibits a molecular weight of 22kDa. GH of animal species is closely homologous in amino acid sequence to that of humans and is therefore very similar in its characteristics.

A major biological effect of GH is to promote growth throughout a range of organs and tissues in the body. GH responsive organs or tissues include the liver, intestine, kidneys, muscles, connective tissue and the skeleton.

Hypopituitary dwarfism is a condition which is readily treated by administering GH to a subject suffering the condition. Prior to the production of large quantities of hGH by recombinant means only limited amounts of hGH could be prepared by laborious extraction of pituitary glands from human cadavers. This practice carried with it risks associated with infectious agents, eg the agent responsible for Creutzfeldt-Jakob disease (CJD), and that these agents might be passed to the patient receiving GH.

The isolation of the hGH gene and the construction of transformed host cells expressing hGH in cell culture has opened up not only a more reliable, safer and more cost effective treatment of hypopituitary dwarfism, but the

possibility of using hGH for treatment of other diseases and conditions as well. A long appreciated problem with aqueous liquid formulations of pharmaceutical proteins, not just hGH, was that of instability during storage over a period of time. hGH in aqueous solution was known to undergo a variety of degradative changes. Chemical changes such as deamidation occur and this may be related to the pH of the solution during storage. Oxidation of methionine residues may occur. There is also the possibility of a clipping of the peptide backbone as a result of hydrolysis. Also, there is the physical change of aggregation, for example, resulting in the formation of opaque insolubles.

Early suggestions about how to solve the problems of instability noted above included freeze drying, but this of course meant that the resulting Iyophilised product needed reconstitution immediately or shortly prior to administration.

In the circumstances of routine self-administration by a patient at home, this normally means that the patient has the task of reconstituting the Iyophilised preparation into an aqueous solution. This is inconvenient for the patient and carries with it a risk of improper reconstitution due to lack of care, lack of attention to detail and instructions, or simply misunderstanding on the part of the patient. Freeze drying of formulations also suffers from the disadvantage of being costly and time consuming from a manufacturing perspective.

WO 89/09614 (Genentech) discloses an hGH formulation intended for Iypohilization prior to reconstitution and administration. The formulation is said to pocess greater stability during processing (including Iypohilization) reconstitution and storage.

Much effort has therefore been expended in finding formulations which permit a simpler self-administration of GH by patients. These efforts have focused on ways of providing sufficiently stable aqueous hGH formulations in a ready to use form.

EP-A-0 131 864 (Hoechst Aktiengesellschaft) describes the prevention of aggregation in proteins of greater than 8.5 kDa in aqueous solution by using surfactants.

EP-A-0 211 601 (International Minerals & Chemical Corporation), although perhaps primarily concerned with sustained release formulations, describes how GH can be stabilised in solution as a liquid by formulating it with nonionic surfactants; in particular, certain polyoxyethylene-polyoxypropylene block copolymers, eg PLURONIC (trade mark of BASF) or GENAPOL (trade mark of Hoechst) block copolymer.

WO 94/03198 (Genentech) is another disclosure following the previous teachings about using non-ionic surfactant as an hGH stabiliser in liquid formulations. The range 0.1-5% (w/v) non-ionic surfactant in the formulation is said to permit the formulation to be exposed to shear and surface stresses without causing denaturation of the GH protein. In particular, the surfactantcontaining formulations are seen as being useful in pulmonary dosing and needle less jet injector guns.

EP-A-0 303 746 (International Minerals and Chemical Corporation) teaches that aqueous GH may be stabilised by formulating it with a polyol, eg nonreducing sugars, sugar alcohols, sugar acids, lactose, pentaerythritol, watersoluble dextrans and Ficoll ; an amino acid, eg glycine, arginine and betaine; an amino acid polymer having a charged side group of physiological pH; and finally a choline derivative, eg choline chloride, choline dihydrogen citrate or dicholine mucate. However, many of the polymeric materials referred to

above may carry some risk in administration to patients. Pharmaceutical regulatory requirements dictate that any unnecessary additives, particularly synthetic additives (eg pentaerythritol) must be avoided in order to reduce risks to patients. Many of the suggested stabilisers in the disclosure would not appear clinically acceptable and therefore would not enable a pharmaceutically acceptable formulation to be made.

WO 92/17200 (Genentech) is concerned with stabilising hGH, not just in liquid but also in Iyophilised preparations. The suggestion is that stable zinc: hGH dimers are produced. The zinc: hGH dimers are made up of two zinc ions and two hGH molecules.

WO 93/12811 (Novo Nordisk) discloses a liquid hGH formulation in which asparagine is used as the stabilising and buffering substance.

WO 93/19776 (Kabi Pharmacia) teaches that when an aqueous hGH product is formulated with citrate buffer then it is more stable than when it is formulated with phosphate buffer.

WO 97/29767 (CSL Limited & Monash University) discloses a method of preparing a stabilised liquid hGH formulation for storage at temperatures not exceeding freezing or refrigeration temperatures.

The present inventors have noticed how crystals tend to form in storagestable aqueous growth hormone formulations known in the art, not just when they are stored at refrigeration temperatures, but also when they are stored above refrigeration temperatures, at least for a time. The presence of crystals in liquid hGH formulations is undesirable because prior to administration such formulations need to be agitated or swirled and there may be instances when crystals are small or unobserved and the formulation is caused to be administered without dissolving the crystals sufficiently first.

There is also the obvious advantage in terms of the visual appearance of hGH formulations when crystals have formed during storage. An object of the invention is therefore to provide liquid hGH formulations which avoid the problem of crystal formation when stored for periods of time, e. g. from about one week to up to 6 or 18 months. Another object of the invention is to provide liquid hGH formulations which exhibit minimal or no crystallisation when stored for at least a period of time outside a refrigerator, e. g. periods of several days, weeks or months.

The present invention therefore provides an aqueous growth hormone formulation comprising growth hormone and: (a) citrate buffer of about pH 5.6 or more, or (b) a buffer other than citrate of about pH 6.0 or more, and substantially free of crystallisation on storage.

Arising out of the present invention the inventors have perceived an advantage for patients, pharmacists and medical practitioners. Hitherto it has been necessary to ensure careful storage of growth hormone formulations at refrigeration temperatures in order to minimize crystallisation.

Prior to receipt of the growth hormone by patients the formulations can usually be reliably stored at refrigeration temperatures (in the range of 40 to 8OC) by manufactures and pharmacists. However, once received and stored by patients in domestic refrigerators there is much less reliability in terms of storage temperature. The present inventors have observed how crystallisation tends to occur more readily at temperatures greater than 8OC, i. e. above refrigeration temperatures.

The formulations of the present invention provide a greater resistance to crystallisation if stored for any time above refrigeration temperatures. This

therefore permits patients to be supplied with sufficient growth hormone to provide daily doses over longer periods of time than was hitherto recommendable or desirable.

Whereas before, patients might have kept a small number of doses for use over a period of a week or weeks, with the formulations of the present invention patients may keep at least one month, possibly two or three months supply of growth hormone in domestic refrigerator with no or only minimal crystallisation taking place. The frequency of prescription to patients can therefore be reduced significantly by the present invention.

In embodiments of the invention employing citrate, this buffer preferably has a pH of no more than about 7.0, or 6.2 i. e. a pH in the range of about 5.6 to about 7.0, more preferably about 5.6 to 6.2, even more preferably a pH of 5.6 or 6.2.

In embodiments of the invention employing a buffer other than citrate then this buffer preferably has a pH of no more than about 7.0 or 6.8, i. e. a pH in the range of about 6.0 to 7.0, more preferably about 6.2 to about 7.0, even more preferably about 6.2 to about 6.8. The buffer other then citrate may be selected from acetate, formate or glycine, preferably sodium acetate or ammonium acetate.

At least some of the overall storage time may be at a temperature above refrigeration temperatures, possibly up to about a week on aggregate, possibly up to about a month on aggregate.

At least a part of the time that the formulation is stored may be at a storage temperature of at least 8OC, optionally a temperature in the range selected from 80 to 400C, 80 to 250C or 80 to 1 5OC.

The formulation is preferably substantially isotonic, more preferably wherein the agent for isotonicity is selected from one or more of a sugar alcohol, a monosaccharide, a disaccharide, propylene glycol or an inorganic salt, even more preferably the agent for isotonicity is selected from one or more of mannitol, lactose, sucrose, propylene glycol, sodium chloride or ammonium chloride.

The formulation preferably further comprises a non-ionic surfactant and/or a preservative. Non-ionic surfactants may include poloxamers, such as poloxamer 184 or 188, Pluronic &commat; polyols, e. g. Pluronic F-68, polysorbates such as polysorbate 20 or 80, for example, and other ethylene/polypropylene block polymers. Amounts used may be in the range from about 0. 1 % (w/v) to about 5% (w/v), more preferably, 0. 1% (w/v) to about 1% (w/v). The preservative may be selected from one or more of phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzylalkonium chloride and benzethonium chloride. Preferred preservatives are phenol at 0. 2-0. 4% (w/v) and benzyl alcohol at 0. 7-1% (w/v).

In preferred formulations the growth hormone is human growth hormone (hGH).

Particularly preferred formulations of the invention are set out below :

Formulation I

hGH 3. 33mg/ml (10 IU/ml) NaH2PO4 1. 05mg/ml (i. e. 1OmM phosphate buffer) Na2HPO4 O. 17mg/ml Mannitol 35mg/ml (3. 5% w/v) Pluronic F-68 2mg/ml (0. 2% w/v)

Phenol 2. 5mg/ml (0. 25% v/v) Water for injection q. s. pH 6. 0 Formulation II hGH 3. 33mg/ml (10 IU/ml) NaH2PO4 1. 05mg/mt (i. e. 10mM phosphate buffer) Na2HPO4 0. 17mg/ml Mannitol 30mg/ml (3. 0% w/v) Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Propylene glycol 2mg/ml (0. 2% v/v) Water for injection q. s. pH 6. 0 Formulation III hGH 3. 33mg/ml (10 IU/ml) Ammonium acetate buffer 10mM Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Ammonium chloride 5mg/ml (0. 5% v/v) Water for injection q. s. pH 6. 0 Formulation IV hGH 3. 33mg/ml (10 IU/ml) Glycine buffer 10mM Mannitol 35mg/ml (3. 5% w/v)

P) uronic F-68 2mg/m) (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 6. 0 Formulation V hGH 3. 33mg/ml (10 IU/ml) Glycine buffer 20mM Mannitol 35mg/ml (3. 5% w/v) Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 6. 0 Formulation V ! hGH 3. 33mg/ml (10 IU/ml) Citrate buffer 11 mM Sodium chloride 5. 9mg/ml (0. 59% w/v) Pluronic F-68 0. 8mg/ml (0. 08% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 5. 6 Formulation VII hGH 3. 33mg/ml (10 IU/ml) Citrate buffer 11mM Sodium chloride 5. 9mg/ml (0. 59% w/v) Pluronic F-68 0. 8mg/ml (0. 08% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v)

Water for injection q. s. pH 6. 0 The present invention also provides a method of inhibiting crystallisation in an aqueous growth hormone formulation comprising formulating the growth hormone in citrate buffer of about pH 5.6 or more, or a buffer other than citrate of about pH 6.0 or more.

Further characteristics of the formulations to be produced in accordance with the methods of the invention are as hereinbefore described.

The present invention also includes the use of an aqueous formulation of growth hormone buffered with: (a) citrate at about pH 5.6 or more, or (b) a buffer other than citrate at about pH 6.0 or more, as a stored pharmaceutical product substantially free of crystallisation.

The invention therefore provides for the use of growth hormone buffered with citrate at pH 5.6 or more, or a buffer other than citrate at about pH 6.0 or more, for the manufacture of a stable aqueous formulation substantially free of crystallisation on storage and for treatment of patients in need of growth hormone.

Prior to storage, hGH formulations normally comprise about 4% of"related proteins"being proteinaceous materials generated by degradative processes of deamidation and oxidation. Such "related proteins" are defined in the European Pharmacopoiea and measured by reversed phase HPLC. The

inventors propose a maximum of 20% "related proteins" as a target at the end of the shelf life of the formulations.

The degradation rate of hGH is not exactly linear and the rate of degradation increases with an increase in temperature. At 2"-8"C formulations usually exhibit an increase in "related proteins" of about 0. 75% per month. At 25 C this rises to 12. 7% per month, and at 400C to about 70% per month. Storage at 25 C for 1 month is approximately equivalent to 17 months storage at 2 - 8 C. Storage at 150C for 1 month is approximately equivalent to 5 months storage at 20'-80C. Continuous storage at a temperature in the range of about 250 to 400C is therefore impractical.

Although the formulations of the present invention offer good resistance to crystallisation even up to 400C, particularly up to 25OC, the rapid formation of "rotated proteins at these temperatures will usually place a more immediate limit on the potential shelf life of formulations.

Rates of "related proteins" formation at different temperatures over time are readily measured by one of average skill and with this information the optimisation and maximum storage time/temperature patterns may be calculated without undue burden. In practice, formulations of the present invention can readily be subjected to a daily rise in temperature slightly above about 8 C due to the opening and closing of a refrigerator door or removal from a refrigerator for periods of an hour or so each day for the purposes administration without significant loss of shelf life. Advantageously, formulations of the present invention would not suffer adversely in terms of degradation or crystallisation if left out of the refrigerator at room temperature for a day or so.

The period of storage may be at least 4 weeks, possibly at least 3 months, or up to 18 months.

The pharmaceutical product preferably comprises at least two, more preferably a multiplicity of doses of growth hormone. The pharmaceutical product is preferably in the form of a container for use with an injection device, e. g. a cartridge for use in a pen injector. The pharmaceutical product may be contained within an injection device, preferably a pen injector.

Further characteristics of the formulations associated with the use aspect of the invention are as hereinbefore described. Growth hormone formulations arising out of the uses of aqueous growth hormone formulations in accordance of the invention include formulations I to VII described above.

The crystallisation which is minimized or avoided in formulations by the present invention appears to be that of growth hormone. Preferably any crystallisation in the liquid formulation is detected directly by eye, more preferably under the light microscope at 5x magnification, even more preferably under the light microscope at 10x magnification. Prior to observation under the light microscope formulations may be filtered and the presence or absence of crystals on the filter determined. When viewing under the light microscope the filter may have a pore size of about Sum.

A particularly preferred test for crystallisation is to store the formulation in a sealed container with no airspace for 3 months at 15 C in the absence of light and then observe the presence or absence of crystals by eye.

The aqueous growth hormone formulations of the present invention are preferably storage stable in the sense that there is no or minimal aggregation of growth hormone during the period of storage. Also, there is preferably no or minimal chemical degradation of growth hormone, e. g. by deamidation. Suitable tests for measuring stability of growth hormone in

aqueous solution are well known in the art e. g. as described in WO 94/03198 (Genentech) incorporated herein by way of reference. In preferred formulations of the present invention, the growth hormone exhibits less than 10% aggregation, preferably less than 1%, more preferably less than 0. 1%, even more preferably less than 0. 01% aggregation.

The invention also provides a cartridge containing any of the liquid formulations as hereinbefore described for use with a pen injector device.

When the subjects for administration are humans then the preferred growth hormone is human growth hormone. Particularly preferred human growth hormone is produced by recombinant means, for example as taught in EP-A0 217 822 (Scios Nova) and incorporated herein by way reference. Variants of human growth hormone which may be used in accordance with the invention, alone or in combination with one another and the native hormone, include the 191 amino acid species known as somatropin and the 192 amino acid N-terminal methionine (met) species known as somatrem. There is also the variant known as hGH-V found naturally in the placenta during pregnancy and for which the gene sequence is known and a recombinant protein has been prepared.

The amount of hGH in the liquid formulation of the invention depends on the volume of the formulation and the number of doses of hGH that volume is intended to provide. A preferred dosage volume is 0. but but volumes in the range 0. 01mil to 1. 0ml may be used. Other preferred dosage volumes may fall in the range 0. 1 mil to 0. 6mi.

In a preferred unit dosage for daily administration the amount of hGH administered is 1.3mg although the precise dosage amount may vary

depending on the particular individual. Dosage amounts in the range 0. 033mg to 3. 33mg hGH may be employed, preferably dosages in the range 0. 33mg to 2. 0mg. Increased dosage amounts are appropriate where the frequency of administration is reduced.

The volumes and/or dosage amounts may vary from individual to individual in accordance with specific advice from the clinician in charge.

Usually, formulations in accordance with the invention may comprise hGH in the range 0. 5mg/ml to 20mg/ml, preferably 1 mg/ml to 15mg/ml, more preferably 2mg/ml to 10mg/ml, even more preferably 3mg/ml to 5mg/ml.

The invention also includes kits comprising an injection device and a separate container containing a liquid growth hormone formulation as hereinbefore described. When the administration device is simply a hypodermic syringe then the kit may comprise the syringe, a needle and a vial or ampoule containing the hGH formulation for use with the syringe. In more preferred embodiments the injection device is other than a simple hypodermic syringe and so the separate container is adapted to engage with the injection device such that in use the liquid formulation in the container is in fluid connection with the outlet of the injection device.

Examples of administration devices include but are not limited to hypodermic syringes and pen injector devices. Particularly preferred injection devices are the pen injectors in which case the container is a cartridge, preferably a disposable cartridge.

In another aspect the invention provides a cartridge containing a liquid growth formulation as hereinbefore described for use with a pen injector device. The cartridge may contain a single dose or multiplicity of doses of growth hormone.

Optionally, in relation to all aspects of the invention, when the buffer other than citrate is phosphate then the formulation pH is not 6. 15 or more. Preferred embodiments of the invention will now be described by way of the following examples.

Example 1 - Prearation and purification of bulk recombinant hGH Recombinant hGH is produced in cell cultures of CHO cells transformed with the hGH gene to express the hGH protein under culture conditions. Details of how the cells are made and grown are described in EP-A-0 217 822 (Scios Nova) incorporated herein by way of reference. The modification of culture conditions for the growth of cultures on an industrial or commercial scale is well within the abilities of one of average skill in the art.

Once produced by the cells in culture, the hGH needs to be extracted and purified into a form suitable for pharmaceutical use. This is carried out according to the procedures described in AU 629177 (University of New South Wales & Garvan Institute of Medical Research) incorporated herein by way of reference. The resultant hGH preparation is in the form of a bulk solution and this is employed in making the formulations described below.

The concentration of hGH in bulk solution was 12. 17mg/ml.

Example 2-Preparation of aqueous growth hormone formulations The formulations were prepared by adding double strength excipient solution to bulk hGH solution which is diluted to give an hGH concentration of about 7mg/ml. The pH of the formulation is then adjusted as required.

The materials used are set out in Table 1 below :

Table 1

Material Quality Supplier Batch NO Somatropin bulk soln Ph Eur 5390008. 1 Sodium Dihydrogen phosphate Ph Eur Merck K24108245 Disodium hydrogen phosphate Ph Eur Merck K21433976 Mannitol Ph Eur Rentschler 9510901 Sucrose Analytical Fluka 365331/112297 Pluronic F-68 USNF Rentschler WE70800665 Benzyl alcohol Ph Eur Weimer WE80800601 Formic acid Analytical Fluka 401203/113399 Acetic acid Ph Eur Merck K23614056651 Ammonium acetate Analytical Fluka 260207688 Ammonium chloride Analytical Fluka 399705/142899 Propylene Glycol Ph Eur Fluka 373212/143397 Glycine Analytical Fluka 345033/1196 Phenol Analytical Fluka 345074/1696 WFI Ph Eur Rentschler 990470 Filter, Millex GV 0. 22SLGV 025BS Millipore R7PM38096 0. 1 M phosphate buffer pH 6.0 was made by mixing 200ml of 0. 1 M disodium phosphate with 43ml of 0. 1 M sodium dihydrogen phosphate. The phosphate salt solutions were prepared as shown in Table 2 below : Table 2

Material Quantity required Quantity added disodium phosphate dihydrate 3. 56 g 3.56 g WFI to 200 mi 200. 03 g sodium dihydrogen phosphate 3. 12 g 3. 12 g dihydrate WFI to 200 ml 200. 06 g 0. 1 M sodium acetate buffer solution was prepared in accordance with Table 3 below :

Table 3

Material Quantity required Quantity added Glacial acetic acid 0. 60g 0. 7 g N NaOH qs to pH 6. 0 12. 1 ml WFI to 100 ml 100. 2g 0. 1M ammonium acetate buffer solution pH 6.0 was made by mixing 1.4ml of 0. 1 M acetic acid with 50g of 0. 1M solution of ammonium acetate in accordance with Table 4 below : Table 4

Material Quantity required Quantity added Glacial acetic acid 0. 60 9 0. 6 9 WFI to 100 mi 100 mol Ammonium acetate 0. 77g 0. 78 g WFI to 100 ml 100 ml 0. 1M formate buffer solution was prepared by mixing components in accordance with Table 5 below : Table 5

Material Quantity required Quantity added Formic acid 0. 46 g 0. 47 g N NaOH qs to pH 6. 0 9.8 ml WFI to 100 ml 100.04 g 0. 1 M glycine buffer solution was prepared by mixing components in accordance with Table 6 below : Table 6

Material Quantity required Quantity added glycine 0. 75 g 0. 75 g N NaOH qs to pH 6. 0 0. 1 ml WFI to 100 ml 100. 05g 20mg/ml Pluronic solution was prepared by mixing components in accordance with Table 7 below :

Table 7

Material Quantity required Quantity added Pluronic F68 5. 0g 5. 01g WFI to 250 ml 250. 19 50g of each of double strength excipient solutions 1-23 were prepared by mixing components as shown in Table 8 below : Table 8

Formulation 1 2 3 4 5 6 7 8 9 10 11 12 Excipient Benzyl alcohol 900 900 900 900 900 900 mg Mannitol mg 350 5000 3500 3500 3500 3500 0 Pluronic F-68 10ml 10ml 10ml 10ml 10ml 10ml 10ml 01ml soltn I Phosphate 10ml 10ml 10ml 10ml l Na buffer 10ml 20ml Formate buffer 10ml 20ml Sucrose 7g 10g Propylene glycol Phenol Glycine WFI to40g40g40 g 55. 3g*40g 40g 40 g 45 g* Divide into 2 # ## # ## # ## # ## # 20g WFI to 50g 25 g 25 g 50 g 27. 06g 27. 33g 50 g 25g 25g 50 g 25g 25g final pH 6.0 5.96 6.15 6.5 * For formulation 5/6 there was difficulty in dissolving the sucrose, for formulation 11 the benzyl alcohol. Therefore they were made to a higher weight than proposed in the protocol. The formate buffer proved difficult to adjust

Table 8 (ctd)

Formulation 13 14 15 16 17 18 19 20 21 22 23 Excipient Benzyl alcohol 900 900 900 900 900 900 Mannitol 3500 35003500 35003000** 3500 3500 Pluronic F-68 soltn 10ml 10ml 10ml 10ml 10ml 10ml 10ml 10 ml Phosphate buffer 10ml 10ml 10ml 20 ml 30 ml Ammon Acetate 10 ml buffer Formate buffer Ammonium 500 Chloride mg Propylene glycol 200 Phenol 250 Glycine 10m 20ml I WFI to 40 g 40 g 40 g 40 g 40 g 40 g 44.2g* 40 g Divide into 2 x 20g ## # ## # ## # WFI to 50g 25g 25g 50g 25g 25g 50g 50g 25g 25g 50 g final pH 5. 98 5. 83 6. 12 6. 20 6. 01 6.20 6.00 6. 01 6.01 6.19 5. 98 *Difficulty in dissolving the Benzyl alcohol ; therefore made to a higher weight.

** Amount of Mannitol reduced from that in the protocol to allow for the tonicity of the propylene glycol.

The glycine buffer proved difficult to adjust Formulations 1-23 were prepared by taking sufficient bulk hGH to give final concentration of hGH of 3. 33mg/ml in 10mi of solution. The hGH concentration in bulk solution was 12. 17mg/ml. 3.28mg/g of hGH in bulk solution was added to a tared 50ml beaker.

By way of calculation :

W= Weight of bulk hGH solution required in grams :- 3. 28x10 X Where X = Concentration of bulk hGH solution in mg/ml (or mg/g) and assuming a density of 1 mg/ml.

W = 2. 70 The bulk hGH solution was diluted to approximately 4,5 ml with WFI.

5g (with the exception of formulation 5 (5. 4g), and formulation 6 (5. 5g) of the double concentrated excipient solution prepared above was then added slowly. The pH was checked and adjusted and the solution increased 10g with WFI.

The 23 formulations were produced in accordance with Table 9 below : Table 9

Formit Weight pH before Solution used and Final pH of bulk adjustme quantity added to adjust nt pH 1 2. 70 6. 07 0. 05 ml 0. 1 M HCl 6.00 2 2. 72 6. 14 0. 1 ml 0. 1M HCI 6. 00 3 2.71 6.27 0.05 ml 0.1M HCl 6.21 4 2.71 6.16 0.1 ml 0.1M HCl 5.98 5 2.70 6.21 0.15 ml 0.1M HCl 5.97 6 2.70 6.29 0.05 ml 0. 1 HCI 6. 23 7 2.70 6.00 - 6.00 8 2.70 5.99 - 5.99 9 2. 71 6. 00 0. 05 ml 0. 1M Nach 6 24 10 10 2.72 5.97 - 5.97 11 68 5.96 - 5.96 12 2.69 6.01 0.05 ml 0.1M NaOH 6.25 13 2. 70 70 5.93 0.05 ml 0.1M NaOH 6.01

Table 9 (ctd)

14 2.68 6.20 0. 2 ml 0. 01M HCI 6.01 15 2.71 5.95 0.05 ml 0. 1M NaOH 6. 23 16 2.70 6.23 0.05 ml 0.1M HCl 6.19 17 2. 70 6. 17 0. 05 mi 0. 1 HCI 6. 02 18 2. 69 6. 26 0. 05 ml 0. 1 M HCl 6.19 19 2. 71 5. 93 0. 05 mi 0. 1M NaOH 6.00 20 2. 72 6. 05 0. 025 ml 0. 1 M HCl 6.01 21 2. 69 6. 08 0. 05 ml 0. 1M HCI 6. 03 22 2.68 6.26 0.05 ml 0.1M HCl 6.22 23 2. 74 5. 87 0. 3 ml 0. 01 M NaOH 6.00 Using a syringe, the solutions were filtered via a 0.22 micron filter into 6 cartridges having the plunger already in place. The seal was crimped in place.

The compositions of the 23 formulations are as shown in Table 10 below :

Table 10

Formulation &num; 1 2 3 4 5 6 7 8 9 10 11 Excipient hGH mg/ml 3. 33 3. 33 3. 33 3. 33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 Benzyl alcohol mg/ml 9.0 9.0 9.0 9.0 9.0 9.0 9. 0 9. 0 9.0 9.0 9.0 Mannitol mg/ml 35. 0 50. 0 50. 0 35. 0 35.0 35.0 35.0 35.0 Pluronic F-68 mg/ml 2. 0 2.0 2.0 2.0 2. 0 2. 0 2.0 2. 0 2.0 2.0 2.0 2.0 Phosphate buffer mM 10 10 10 10 10 10 Na Acetate buffer mM 10 20 20 Formate buffer mM 10 20 Sucrose mg/ml 70 100 100 pH 6 6 6. 2 6 6 6. 2 6 6 6. 2 6 6 Fonnulation 12 13 14 15 16 17 18 19 20 21 22 23 Excipient hGH mg/ml 3.33 3.33 3.33 3. 33 3. 33 3.33 3.33 3.33 3.33 3.33 3.33 3.33

Table 10 (ctd)

Benzyl alcohol mg/ml 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 Mannitol mg/ml 35.0 35.0 35.0 35.0 35. 0 35.0 35.0 30.0 35. 0 35. 0 35. 0 Pluronic uronic F-68 mg/ml 2.0 2.0 2. 0 2.0 2. 0 2. 0 2. 0 2.0 2.0 2.0 2. 0 2. 0 2. 0 Phosphate buffer mM 10 10 10 10 20 30 30 Amm Ac buffer mM 10 Formate buffer mM 20 Propylene glycol 2 mg/ml Phenol mg/ml 2. 5 2. 5 Glycine mg/ml 10 20 20 Ammonium Chloride 5. 0 mg/ml pH 6. 2 6 6 6.2 6. 2 6 6.2 6 6 6 6. 2 6. 0

Examno/e 3-Storaqe of formulations 1-23 and assessment of -ge of f crystallisation For each of formulations 1-23, two cartridges were stored at 2-8OC, two at 15 C and two at 25OC. The cartridges stored at 15"and 25 C were examined by eye for the presence or absence of crystals at frequent intervals.

Table 11 below shows the results for formulations at pH 6. 0. The shaded boxes show formulations where crystallisation was observed.

Table 11

Time weeks Formulation 13 p 1 Phos, 15 C 25 C 2 Phos, man, plur, BzOH 6.0 15 C 25 C 20 Phos, man, plur, BzOH 6.0 15 C 25 C 21 Phos, man, plur, BzOH 15 C 25 C 17 15 C 25 C 19 plur, BzOH, propgly 6.0 15 C 25 C 4 Phos, suc, plur, BzOH 6.0 15 C 25 C 5 Phos, suc, plur, BzOH 6.0 15 C 25 C 7 NaAc, man, plur, BzOH 6.0 15 C 25 C 8 NaAc, man, plur, BzOH 6.0 15 C 25 C 10 15 C 25 C 11 plur, BzOH 6.0 15 C 25 C 13 Glyc, man, plur, BzOH 6.0 15 C 25 C 14 Glyc, man, plur, BzOH 6.0 15 C 25 C 23NH4Ac, NH4cl, plur, BzOH 6.0 15 C 25 C

Formulations 17, 19 and 23 at pH 6. 2 are most recommendable to avoid crystallisation on storage above refrigeration temperatures. Formulations 13 and 14 are also recommendable but the glycine buffer is probably outside its reliable range at pH 6. 0.

None of the formulations at pH 6. 2 or above showed any crystallisation during the 3 months of study, whether stored at 150 or 25OC.

Example 4-Preparation and test storage offbnu/a/ons 24-38 ie of formulations 24-38 Formulations 24-38 were prepared having the compositions shown in Table 12 below : Table 12

Formulation N 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Ingredient (mg/ml) 3.33 3.33 3.33 3.33 3.33 3.33 3.33 3.33 Pluronic 0.8 - 0.8 - - - 0.8 - 0.8 1.6 3 0.8 0.8 0.8 Citric acid H2O 1.8 1.8 1.8 1 1 - 1 0.3 0.3 0.5 0.3 0.3 0.4 0.5 1.8 Trisodium 2.5 Sodium 5.9 Benzyl alcohol 9 9 9 - - - - 9 9 - 9 9 9 9 9 Phenol - - - 2.5 - - - - m-cresol - - - - - - 3 - - - - - - - Na2HPO4 - - - - - 0.5 - - - - - - - - NaH2PO4 - - - - - 2.8 - - - - - - - - pH 6.0 6.0 None of formulations 24-38 exhibit crystallisation when stored at 25 C for 3 months.

Claims

Claims 1. An aqueous growth hormone formulation comprising growth hormone and: (a) citrate buffer of about pH 5.6 or more, or (b) a buffer other than citrate of about pH 6.0 or more, and substantially free of crystallisation on storage.
2. A formulation as claimed in claim 1, wherein at least some of the storage time is at a temperature above refrigeration temperatures.
3. A formulation as claimed in claim 1 or claim 2, wherein storage is for at least a week, preferably at least 4 weeks, more preferably at least 3 months.
4. A formulation as claimed in any of claims 1 to 3, wherein the citrate buffer has a pH in the range of about 5.6 to about 7.0, preferably about 5.6 to about 6.2, more preferably a pH of 5.6 or 6.2.
5. A formulation as claimed in any of claims 1 to 4, wherein the buffer other than citrate has a pH in the range of about 6.0 to 7.0, preferably about 6.2 to about 7.0, more preferably about 6.2 to about 6.8.
6. A formulation as claimed in any of claims 1 to 3 or 5, wherein the buffer other than citrate is phosphate, optionally a buffer selected from acetate, formate or glycine, preferably sodium acetate or ammonium acetate.
7. A formulation as claimed in any preceding claim, wherein the storage temperature is at least 8OC, optionally a temperature in a range selected from 80 to 25OC or 80 to 15OC.
8. A formulation as claimed in any preceding claim, wherein the formulation is substantially isotonic, preferably wherein the agent for isotonicity is selected from one or more of a sugar alcohol, a monosaccharide, a disaccharide, propylene glycol or an inorganic salt, more preferably the agent for isotonicity is selected from one or more of mannitol, lactose, sucrose, propylene glycol, sodium chloride or ammonium chloride.
9. A formulation as claimed in any preceding claim, further comprising a non-ionic surfactant and/or a preservative, optionally wherein the non-ionic surfactant is Pluronic F-68, optionally wherein the preservative is selected from one or more of phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzylalkonium chloride and benzethonium chloride.
10. A formulation as claimed in any preceding claim, wherein the growth hormone is human.
11. A formulation as claimed in any preceding claim selected from :

Formulation I hGH 3. 33mg/ml (10 IU/ml) NaH2PO4 1. 05mg/mt (ie 10mM phosphate buffer) Na2HPO4 0. 17mg/ml Mannitol 35mg/ml (3. 5% w/v) P) uronic F-68 2mg/m) (0. 2% w/v) Phenol 2. 5mg/ml (0. 25% v/v) Water for injection q. s. pH 6. 0

Formulation 11 hGH 3. 33mg/ml (10 IU/ml) NaH2PO4 1. 05mg/m) (ie 10mM phosphate buffer) Na2HPO4 0. 17mg/ml Mannitol 30mg/ml (3. 0% w/v) Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Propylene glycol 2mg/ml (0. 2% v/v) Water for injection q. s. pH 6. 0 Formulation III hGH 3. 33mg/ml (10 IU/ml) Ammonium acetate buffer 10mM Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Ammonium chloride 5mg/ml (0. 5% v/v) Water for injection q. s. pH 6. 0 Formulation IV hGH 3. 33mg/ml (10 IU/ml) Glycine buffer 10mM Mannitol 35mg/ml (3. 5% w/v) Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s.

pH 6. 0 Formulation V hGH 3. 33mg/ml (10 IU/ml) Glycine buffer 20mM Mannitol 35mg/ml (3. 5% w/v) Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 6. 0 Formulation VI hGH 3. 33mg/ml (10 IU/ml) Citrate buffer 11mM Sodium chloride 5. 9mg/ml (0. 59% w/v) Pluronic F-68 0. 8mg/ml (0. 08% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 5. 6 Formulation VII hGH 3. 33mg/ml (10 IU/ml) Citrate buffer 11mM Sodium chloride 5. 9mg/ml (0. 59% w/v) Pluronic F-68 0. 8mg/ml (0. 08% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 6. 0
12. A method of inhibiting crystallisation in an aqueous growth hormone formulation comprising formulating the growth hormone in citrate buffer of about pH 5.6 or more, or a buffer other than citrate of about pH 6.0 or more.
13. A method as claimed in claim 12, wherein the citrate buffer has a pH in the range of about 5.6 to about 7.0, preferably about 5.6 to about 6.2, more preferably a pH of 5.6 or 6.2.
14. A method as claimed in claim 12 or claim 13, wherein the buffer other than citrate has a pH in the range of about 6.0 to 7.0, preferably about 6.2 to about 7.0, more preferably about 6.2 to about 6.8.
15. A method as claimed in any of claims 12 to 14, wherein the buffer other than citrate is phosphate, optionally a buffer selected from acetate, formate or glycine, preferably sodium acetate or ammonium acetate.
16. A method as claimed in any of claims 12 to 15, further including formulating the growth hormone to be substantially isotonic, preferably wherein the agent for isotonicity is selected from one or more of a sugar alcohol, a monosaccharide, a disaccharide, propylene glycol or an inorganic salt, more preferably the agent for isotonicity is selected from one or more of mannitol, lactose, sucrose, propylene glycol, sodium chloride or ammonium chloride.
17. A method as claimed in any one of claims 12 to 16, further including formulating the growth hormone with a non-ionic surfactant and/or a preservative, optionally wherein the non-ionic surfactant is a Pluronic F-68, optionally wherein the preservative is selected from one or more of phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzylalkonium chloride and benzethonium chloride.
18. A method as claimed in any of claims 12 to 17, wherein the growth hormone is human.
19. The use of an aqueous formulation of growth hormone buffered with: (a) citrate at about pH 5.6 or more, or (b) a buffer other than citrate at about pH 6.0 or more, as a stored pharmaceutical product substantially free of crystallisation.
20. A use as claimed in claim 19, wherein the product is stored for at least some of the time above refrigeration temperatures, e. g. above 8 C.
21. A use as claimed in claim 20, wherein the product is stored above refrigeration temperatures, e. g. above 8 C, all of the time.
22. A use as claimed in any of claims 19 to 21, wherein the period of storage is at least 4 weeks, preferably at least 3 months.
23. A use as claimed in any of claims 19 to 22, wherein the citrate buffer has a pH in the range of about 5.6 to about 7.0, preferably about 5.6 to about 6.2, more preferably a pH of 5.6 or 6.2.
24. A use as claimed in any of claims 19 to 22, wherein the buffer other than citrate has a pH in the range of about 6.0 to 7.0, preferably about 6.2 to about 7.0, more preferably about 6.2 to about 6.8.
25. A use as claimed in any of claims 19 to 22 or 24, wherein the buffer other than citrate is phosphate, optionally a buffer selected from acetate, formate or glycine, preferably sodium acetate or ammonium acetate.
26. A use as claimed in any of claims 19 to 25, wherein the storage temperature is in the range 80 to 25OC, preferably 80 to 15OC.
27. A use as claimed in any of claims 19 to 26, wherein the formulation is substantially isotonic, preferably wherein the agent for isotonicity is selected from one or more of a sugar alcohol, a monosaccharide, a disaccharide, propylene glycol or an inorganic salt, more preferably the agent for isotonicity is selected from one or more of mannitol, lactose, sucrose, propylene glycol, sodium chloride or ammonium chloride.
28. A use as claimed in any of claims 19 to 27, further comprising a nonionic surfactant and/or a preservative, optionally wherein the non-ionic surfactant is Pluronic F-68, optionally wherein the preservative is selected from one or more of phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, benzylalkonium chloride and benzethonium chloride.
29. A use as claimed in any of claims 19 to 28, wherein the growth hormone is human.
30. A use as claimed in any of claims 19 to 29, wherein the pharmaceutical product comprises at least two, preferably a multiplicity of doses.
31. A use as claimed in any of claims 19 to 30, wherein the pharmaceutical product is in the form of a container for use with an injection device, e. g. a cartridge for use in a pen injector.
32. A use as claimed in any of claims 19 to 31, wherein the pharmaceutical product is contained in an injection device, preferably a pen injector.
33. A use as claimed in any preceding claim, wherein the formulation is :

Formulation I hGH 3. 33mg/m ! (10iU/m !) NaH2PO4 1. 05mg/ml (ie 10mM phosphate buffer) Na2HPO4 0. 17mg/ml Mannitol 35mg/ml (3. 5% w/v) Pluronic F-68 2mg/ml (0. 2% w/v) Phenol 2. 5mg/ml (0. 25% v/v) Water for injection q. s. pH 6. 0 Formulation il hGH 3. 33mg/ml (10 IU/ml) NaH2PO4 1. 05mg/mt (ie 1OmM phosphate buffer) Na2HPO4 0. 17mg/ml Mannitol 30mg/ml (3. 0% w/v) P) uronic F-68 2mg/m) (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Propylene glycol 2mg/ml (0. 2% v/v) Water for injection q. s. pH 6. 0 Formulation III hGH 3. 33mg/ml (10 IU/ml) Ammonium acetate buffer 10mM

Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Ammonium chloride 5mg/ml (0. 5% v/v) Water for injection q. s. pH 6. 0 Formulation IV hGH 3. 33mg/ml (10 IU/ml) Glycine buffer 10mM Mannitol 35mg/ml (3. 5% w/v) Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 6. 0 Formulation V hGH 3. 33mg/ml (10 IU/ml) Glycine buffer 20mM Mannitol 35mg/ml (3. 5% w/v) Pluronic F-68 2mg/ml (0. 2% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 6. 0 Formulation VI hGH 3. 33mg/ml (10 IU/ml) Citrate buffer 11mM

Sodium chloride 5. 9mg/ml (0. 59% w/v) Pluronic F-68 0. 8mg/ml (0. 08% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 5. 6 Formulation VII hGH 3. 33mg/ml (10 IU/ml) Citrate buffer 11mM Sodium chloride 5. 9mg/ml (0. 59% w/v) Pluronic F-68 0. 8mg/ml (0. 08% w/v) Benzyl alcohol 9mg/ml (0. 9% v/v) Water for injection q. s. pH 6. 0
34. A kit comprising an injection device and a container of an aqueous growth hormone formulation as claimed in any of claims 1 to 11.
35. An injection device loaded with an aqueous growth hormone formulation of any of claims 1 to 11.