There are always "fillers" in a stable pharmaceutical preparation. Here is an example of one such formula and the reasons for the various components

The formulation of the subject invention comprises:

a) hGH

b) Glycine

c) Mannitol

d) Buffer

wherein the molar ratio of hGH:glycine is 1:50-200, advantageously 1:75-125, and the molar ratio of hGH:mannitol is 1:700-3000, advantageously 1:800-1500. In a preferred embodiment the buffer is a phosphate buffer and the molar ratio of hGH:phosphate buffer is 1:50-250, advantageously 1:75-150. In another embodiment a non-ionic surfactant is added to the formulation. Advantageously polysorbate 80 is used, and the molar ratio of hGH:polysorbate 80 is 1:0.07.30, advantageously 1:0-1.10.

In a preferred embodiment, the formulation of the subject invention comprises the following components at pH 7.4:

______________________________________ Quantity per ml uponIngredient reconstitution (mg) Molar Ratio______________________________________r-hGH 1.0 1Glycine 0.34 100Mannitol 9.0 1100 NaH2 PO4.H2 O 0.18 110Na2 HPO4.12H2 O 1.32Polysorbate 80 0.20 3______________________________________

In general, the formulations of the subject invention may contain other components in amounts preferably not detracting from the preparation of stable forms and in amounts suitable for effective, safe pharmaceutical administration.

Suitable pH ranges, adjusted with buffer, for the preparation of the formulations hereof are from about 4 to about 8, advantageously about 6 to about 8, most advantageously 7.4. The formulation pH should be less than 7.5 to reduce deamidation (see FIG. 1). pH values below 7.0 result in particulate formation upon lyophilization (see FIG. 2). The aggregation is not related to deamidation.

Storage of lyophilized r.hGH at 40 and 60° C. resulted in increased formation of a trailing peak by HPLC. This peak increased with lower pH values (see FIGS. 3 and 4). Consequently pH 7.4 is an advantageous pH.

The molar ratio of hGH:glycine is 1:50-200, advantageously 1:75-125, most advantageously 1:100. Glycine greatly inhibits dimer formation when it is added in these ratios. Ratios of 1:10 and 1:1000 result is substantial dimer formation upon lyophilization. Glycine, which is a nonessential amino acid, has the formula NH2CH2 COOH. In addition to glycine, an amino acid such as alanine or derivatives of such amino acids are used in the subject formulation.

The molar ratio of hGH:mannitol is 1:700-2000, advantageously 1:800-1500, and most advantageously 1:1100. A formulation containing mannitol as the sole bulking agent, results in greater aggregate and dimer formation than one containing a mixture of mannitol and glycine. As an alternative to mannitol, other sugars or sugar alcohols are used such as sucrose, maltose, fructose, lactose and the like.

The preferred buffer is a phosphate buffer and the molar ratio of hGH:phosphate buffer is 1:50-250, advantageously 1:75-150, most advantageously 1:110. A buffer concentration greater than or equal to 2.5mM and less than 20mM is preferred, most advantageously 5-10mM (see FIG. 5). In this concentration range of buffer, minimal aggregation occurs. Advantageously a sodium phosphate or tris buffer is used.

The effect of using a mannitol-glycine mixture as the lyophilization bulking matrix is compared with using either mannitol alone, or glycine alone in FIGS. 6 and 7. All samples were buffered with 5 mM sodium phosphate buffer, pH 7.4. These figures are plots of the influence of bulking matrix on the formation of dimer over time, at a storage temperature of 40° C.

FIG. 6 demonstrates that a molar ratio of hGH:glycine:mannitol of 1:100:1100 results in the formation of less dimer upon storage, than either mannitol alone or glycine alone.

The importance of the molar ratio of hGH to glycine is shown in FIG. 7, wherein the hGH:mannitol molar ratio is fixed at 1:1100, and the hGH:glycine molar ratio is varied from 1:10, 1:100, 1:1000. The least amount of dimer forms in the sample which has an hGH:glycine molar ratio of 1:100. More dimer is formed in the other two cases.

The formulation of the subject invention may optionally include one of several types of non-ionic surfactants, such as the polysorbates (e.g. polysorbate 20, 80, etc.) and the poloxamers (e.g. poloxamer 188). When polysorbate 80 is used the molar ratio of hGH:polysorbate 80 is 1:0.07-30, advantageously 1:0.1.10, and most advantageously 1:3. On a weight to volume basis, polysorbate 80 is added in amounts of about 0.001 to about 2% (w/v), in order to enhance further the stability of the hGH. Polysorbate 80, in concentrations above 0.01% (w/v) reduces the amount of aggregation forming upon lyophilization. In addition to improved shelf life, the surfactant containing formulation of the subject invention inhibits the formation of protein aggregates when the reconstituted formulation is shaken.

Other pharmaceutically acceptable excipients well known to those skilled in the art may also form a part of the subject compositions. These include, for example, various bulking agents, additional buffering agents, chelating agents, antioxidants, preservatives, cosolvents, and the like., specific examples of these could include, trimethylamine salts ("Tris buffer"), and disodium edetate. In one embodiment, no proteins other than hGH are part of the formulation.

In a further embodiment of this invention, the use of nonionic surfactants permits the formulation to be exposed to shear and surface stresses without causing denaturation of the protein. Further, such surfactant containing formulations, may be employed in aerosol devices such as those used in a pulmonary dosing, and needleless jet injector guns.

In order to prevent surface induced denaturation of hGH that occurs during aerosolization of an hGH formulation concentrations of non-ionic surfactants in the range 0.1-5% (w/v) are used. FIG. 8A shows the severe aggregation of hGH in a mannitol/phosphate buffer upon aerosolization. Only about 30% of the protein is present as intact monomer. The remainder has formed dimer. trimer and higher order aggregates. The formation of aggregates was eliminated as shown in FIG. 8B which was obtained from a sample after aerosolization of the hGH in a mannitol phosphate buffer, containing 1% polysorbate 80.