HK1175711B - Formulation for hgh and rhigf-1 combination - Google Patents

Description

hGH and rhIGF-1 combination formulations

The present invention relates to pharmaceutical compositions. More particularly, the invention relates to the formulation of Growth Hormone (GH) and insulin-like growth factor (IGF-1) combination compositions. These combination compositions provide stable liquid pharmaceutical compositions at the desired pH without the formation of visible insoluble aggregates.

The invention further provides formulations of insulin-like growth factor 1(IGF-1) and Growth Hormone (GH), wherein the proteins may be formulated together in injectable form, or separately and mixed into unit dose injectable form prior to administration.

Insulin-like growth hormone belongs to a family of polypeptides known as growth regulators and is a polypeptide naturally present in human body fluids. Most tissues, and in particular the liver, produce IGF-1 with specific IGF-binding proteins. IGF-1 stimulates the growth and division of a variety of cell types, particularly during development, and thus processes such as bone growth and cell replication are affected by IGF-1 levels. These molecules are all controlled by Growth Hormone (GH).

IGF-1 is the major protein hormone mediating the growth promoting effects of GH on bone. IGF-1 is produced in response to GH and then induces subsequent cellular responses, including cellular responses in bone. IGF-1 is a single chain of 70 amino acids with 3 intramolecular disulfide bonds. IGF-1 has a molecular weight of 7649 daltons, and is produced primarily by the liver as an endocrine hormone, and in a paracrine/autocrine fashion in the target tissues. IGF-1 has been produced recombinantly in large scale using yeast and E.coli (E.coli) (rhIGF-1).

Growth hormone or human growth hormone (hGH) is a single chain polypeptide consisting of 191 amino acids. Disulfide bonds are linked at positions 53 and 165 and at positions 182 and 189. Human GH is a potent anabolic agent (anabolic agent). Among these, the most dramatic effect in hypopituitary (GH-deficient) individuals is the acceleration of linear growth of bone growth plate cartilage leading to increased height.

The international patent application WO9118621 describes the advantages and synergy of a combination of two proteins. Co-administration of IGF-1 and GH to a mammal results in an increase in growth over that achieved using IGF-1 or GH alone. The growth is equal to the sum of the growth observed when IGF-1 is administered and the growth observed when GH is administered.

Methods and compositions for increasing growth rate are also disclosed in international patent application WO 2006/130769. The study was primarily directed to treatment methods and results focused on patient response. Pharmaceutical compositions, and in particular mixtures of IGF-1 and GH formulated in mannitol, glycine and/or phosphate at pH 7.4 are described. If the mixture is to be stored, it is formulated in a buffer, e.g., a citrate salt at a pH of about 6 containing a surfactant that increases GH solubility at that pH, e.g., polysorbate 20 or poloxamer 188. It also describes the feasibility of adding inorganic salts and stabilizers. No non-aggregating agent is used in the formulation disclosed in WO 2006/130769.

A problem that often arises when combining two proteins in solution is the formation of complexes due to protein-protein interactions. The formation of such complexes is particularly affected by variations in concentration, temperature, pH and buffer in the protein-containing solution. The protein complex may then form insoluble aggregates that cause a loss of protein potency and activity.

Furthermore, in pharmaceutical formulations, the dosage of the therapeutic protein is important and must be preserved for a long period of time within a controlled range. The use of solubilizing agents is often required to obtain and maintain a suitable concentration of protein in solution, and in particular to solubilize large amounts of protein. U.S. patent 6,767,892 discloses pharmaceutical compositions of IGF-1 and its analogs with solubilizing compounds such as arginine, N-acetyl arginine or guanidine hydrochloride IGF-1. The compositions were tested and the comparative data showed an increase in solubility of IGF-1 at pH values greater than 5.0 and at refrigeration temperatures. However, this document does not disclose a composition comprising IGF-1 in combination with a further therapeutic protein.

It is an object of the present invention to prepare a liquid formulation comprising IGF-1 and Growth Hormone (GH) which is stable at 4 ℃ for at least 30 days without significant aggregation as evidenced by visual clarity of the solution. A further object of the invention is a process for the preparation of a liquid formulation comprising IGF-1 and GH.

Drawings

FIG. 1: showing overlapping sedimentation velocity profiles obtained by analytical ultracentrifugation of IGF-1 solution, GH solution and 1:1 mixture of the two solutions. The first set of curves (fig. 1) was obtained with proteins formulated in 25mM citrate buffer at pH6 and showed evidence of a substantial correlation between the proteins.

FIG. 2: the sedimentation curve of a solution containing 100mM arginine ion (arginine) is shown. The curve shows that the presence of arginine produces a change indicating a decrease in the amount of high molecular weight aggregates in solution.

The following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention.

According to the present invention, the term "non-aggregating agent" relates to a compound that prevents or reduces the formation of insoluble protein aggregates when the protein is placed in solution.

The term "IGF-1" refers to insulin-like growth factor-1 from any species, including but not limited to bovine, ovine, porcine, avian and preferably human, native sequence or variant forms, as well as from any source, naturally synthesized or recombinant.

Preferably, IGF-1 is as e.g. US 6,331,414Said recombinant is produced. More preferably, IGF-1 is the commercial product INCELEX^TMThe active pharmaceutical ingredient of (1).

The term "rhIGF-1" refers to recombinant human IGF-1.

The term "GH" refers to a growth hormone derived from any species, including but not limited to bovine, ovine, porcine, avian and preferably human, in its native sequence or variant form, as well as from any source, naturally synthesized or recombinant.

The terms "human growth hormone" and "hGH" relate to human growth hormone produced by methods including extraction and purification of natural origin and by recombinant cell culture systems, for example, the scientific publication "Direct expression in Escherichia coli of DNA sequence coding for human growth hormone (Direct expression of DNA sequence coding for human growth hormone in E.coli)" Goeddel & al, Nature Vol.281, month 10 1979. The sequence of hGH has been elucidated, for example, in Hormon Drugs, Gueriguian et al, USP convergence, Rockville, Md (1982). The term also includes biologically active human hormone equivalents, e.g., comprising one or more different amino acids in the overall sequence. Furthermore, the terms used in the present application are intended to include amino acid variants of the hGH, i.e. one or more analogs and/or homologs of the hGH which are post-translationally modified, by substitution, deletion and insertion. The two species commonly used are the 191 amino acid natural species (growth hormone) and the 192 amino acid N-terminal methionine species, both obtained recombinantly.

Preference is given to using methionyl human growth hormone (met-hGH) produced in E.coli, under the trade mark by Genentech, IncSold and identical to the native polypeptide except for the presence of the N-terminal methionine residue. Also preferred is available from Genentech, incRecombination of (a) hGH. More preferably, it is available from Genentech, incThe recombinant rhGH injectable liquid of (1).

The term "buffer" as used herein refers to a pharmaceutically acceptable buffer preferably having a pH of 5 to 6.5. Suitable buffers include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, succinate buffers, and amino acid buffers, such as histidine buffers and all salts thereof.

The term "preservative" as used herein means a pharmaceutically acceptable substance that prevents decomposition by microbial growth or undesirable chemical changes.

The term "surfactant" as used herein means a pharmaceutically acceptable substance that is dispersed or suspended by lowering the surface tension of a solvent (e.g., water) or the interfacial tension between two immiscible liquids. Suitable surfactants are, for example, nonionic surfactants, such as polysorbates or poloxamers.

The term "bulking agent" as used herein means a pharmaceutically acceptable substance used to increase the amount of solids, and such as sucrose, trehalose and mannitol, but is not limited to those listed.

The term "tonicity modifying agent" refers to an isotonic modifying agent or osmotic pressure regulating substance that provides osmolality to a buffered solution. Osmolality refers to the total osmotic activity provided by ionic and non-ionic molecules to a solution comprising inorganic salts such as sodium and potassium chloride, polyethylene glycol (PEG), polypropylene, glycols, glycine, glycerol.

The term "lyophilization" as used herein refers to a formulation that is subjected to a process known in the art as lyophilization, which comprises freezing the formulation and subsequently removing ice from the frozen contents.

The term "amino acid" as used herein denotes an amino acid (a free amino acid, i.e. an amino acid that is not in a polypeptide or protein sequence). Amino acids as used herein include, but are not limited to, for example, arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tryptophan, serine, methionine, and proline.

The term "IRF" or "immediate release formulation" refers to a pharmaceutical composition or mixture of pharmaceutical compositions, preferably in liquid form, wherein the carrier which modulates the bioavailability of the pharmaceutically active substance in the tissue is not present at the site of drug administration in the patient's body.

The term "non-aggregating agent" as used herein refers to a product that prevents proteins from interacting to form complexes and/or aggregates when mixed together in solution.

According to the invention, the pharmaceutical composition comprises rhIGF-1 and rhGH as well as

A non-aggregating agent;

a buffer;

a surfactant;

optionally a preservative; and

optionally a tonicity modifier or filler.

Wherein the non-aggregating agent is present in the composition at a concentration of at least 80 mM.

The pharmaceutical composition of the present invention is characterized in that the two active ingredients IGF-1 and GH are present in a single formulation. As used herein, "single formulation" is also referred to as "co-formulation" or "blend (co-mix)". The terms co-formulation or blend are used interchangeably herein.

Preferably, the two active ingredients are human IGF-1 and GH, also referred to herein as hIGF-1 and hGH. It is further preferred that both active ingredients are produced by recombinant means.

In a preferred embodiment, the pharmaceutical composition of the invention is a liquid composition. It is further preferred that it is a multi-dose composition. In embodiments of multi-dose compositions, a preservative is preferably present.

In a further aspect, the present invention relates to a process for the preparation of a pharmaceutical composition comprising IGF-1 and GH. One method of the invention for preparing a pharmaceutical composition may be carried out as follows:

a) preparing a hGH solution in a buffer comprising a non-aggregating agent, a tonicity modifying agent or a bulking agent at a pH of 5 to 6.5;

b) preparing an IGF-1 solution by dialyzing the IGF-1 preparation into the buffer used in step (a) comprising the non-aggregating agent and the tonicity modifying agent or bulking agent;

c) adding a surfactant and optionally a preservative to both stock solutions; and

d) the hGH solution and IGF-1 solution were mixed together.

In an embodiment of the method, in step (a) the lyophilized hGH is dissolved in a buffer or the liquid hGH (e.g. about 20mg/mL solution in bicarbonate buffer) is buffer exchanged into another buffer suitable for pH, preferably about 5 to 6.5, preferably a citrate, succinate or histidine buffer comprising a non-aggregating agent at a concentration in the range of 80 to 200mM, preferably in the range of about 100mM to about 150 mM. Optionally, the at least one solution prepared in any of steps (a), (b), (c) or (d) comprises a preservative, preferably phenol or benzyl alcohol.

In the context of the amount of an ingredient present herein, the term "about" means that the amount may vary by less than ± 20%, or less than ± 15%, or less than ± 10%, or less than ± 5%.

In step (b), lyophilized IGF-1 is dissolved in a buffer, or liquid IGF-1 (e.g., about 20-35mg/mL in citrate buffer) is buffer exchanged into another buffer, preferably citrate, succinate or histidine, at a suitable pH, preferably about 5 to 6.5, which contains a non-aggregating agent at a concentration ranging from about 80 to about 200 mM.

Then, the two solutions prepared separately were mixed together.

Alternative methods for preparing pharmaceutical compositions are also encompassed by the present invention.

According to the present invention, an alternative process for preparing the pharmaceutical composition of the present invention comprises:

a) preparing a solution I having or adjusted to a pH of about 5.8 by mixing a buffer, preferably a histidine buffer, a non-aggregating agent, preferably arginine, a surfactant, preferably polysorbate 20, optionally a preservative, preferably benzyl alcohol, and optionally adjusting the volume with water;

b) preparing an IGF-1 solution in the buffer and non-aggregating agent used in step a) to obtain a solution II;

c) adding the solution II into the solution I to obtain a solution III;

d) preparing a solution IV having or adjusted to a pH of about 5.8 by mixing a buffer, preferably histidine, a non-aggregating agent, preferably arginine, a surfactant, preferably polysorbate 20, optionally a preservative, preferably benzyl alcohol, and optionally adjusting the volume with water;

e) preparing a solution of GH, optionally comprising a sodium bicarbonate buffer, in the buffer and non-aggregating agent used in step d), to obtain solution V;

f) adding the solution V into the solution IV to obtain a solution VI;

g) optionally, filtering solutions III and VI independently;

h) mixing the filtered solutions III and VI in a ratio of IGF-1: GH (w/w) of about 1:1 to 7:1(w/w), preferably 1.1:1(w/w), 3.3:1(w/w) and 6.6:1 to give a solution VII; and

i) optionally, the solution VII is filtered.

Steps (b) and (e) may be performed, for example, by: the solution comprising IGF-1 or GH is diafiltered into a suitable buffer and non-aggregating agent or any other suitable solution to give solutions II and IV.

In one embodiment, solution I and solution IV are the same. In this embodiment, step (d) is absent, i.e. solution IV is not prepared. Solution V was simply mixed with solution I to give solution VI.

In one embodiment, solutions II and IV may comprise bulking agents, such as sucrose or mannitol.

In one embodiment, the liquid GH drug substance (i.e. the solution comprising GH, preferably hGH and more preferably rhGH) is mixed directly with solution IV without any prior buffer exchange or penetration into buffers and non-aggregating agents according to step e), i.e. without performing step e) as described above.

Thus, in this embodiment, the method comprises the steps of:

a) preparing a solution I having or adjusted to a pH of about 5.8 by mixing a buffer, preferably a histidine buffer, a non-aggregating agent, preferably arginine, a surfactant, preferably polysorbate 20, optionally a preservative, preferably benzyl alcohol, and optionally adjusting the volume with water;

b) preparing an IGF-1 solution in the buffer and non-aggregating agent used in step a) to obtain a solution II;

c) adding the solution II into the solution I to obtain a solution III;

d) preparing a solution IV having or adjusted to a pH of about 5.8 by mixing a buffer, preferably histidine, a non-aggregating agent, preferably arginine, a surfactant, preferably polysorbate 20, optionally a preservative, preferably benzyl alcohol, and optionally adjusting the volume with water;

e) -alternative embodiments: adding GH drug substance, optionally comprising sodium bicarbonate buffer, to solution IV to give solution VI;

f) optionally, filtering solutions III and VI independently;

g) mixing the filtered solutions III and VI in a ratio of IGF-1: GH (w/w) of about 1:1 to 7:1(w/w), preferably 1.1:1(w/w), 3.3:1(w/w) and 6.6:1 to give a solution VII; and

h) optionally, the solution VII is filtered.

In an embodiment of this variant, solution I and solution IV are identical. In this embodiment, step (d) is absent, i.e., solution IV is not prepared, and the GH drug substance is simply mixed with solution I to give solution VI.

Preferably, the liquid hGH drug substance is an about 20mg/mL hGH solution in bicarbonate buffer at a concentration of about 6-10mM, preferably 7.5mM, and diluted without diafiltration to a suitable pH, preferably about 5 to 6.2, preferably citrate, succinate or histidine, and optionally containing a non-aggregating agent at a concentration in the range of about 80 to 200mM, preferably about 100mM or about 150 mM.

In another embodiment, liquid IGF-1 (e.g., about 20-35mg/mL in 200mM citrate buffer) is buffer exchanged into another buffer suitable for pH, preferably about 5 to 6.5, preferably a citrate, succinate or histidine buffer, optionally containing a non-aggregating agent at a concentration ranging from about 80 to about 200mM, preferably from about 100mM to about 150 mM. The two separately prepared solutions were then mixed together.

The filtration may be performed by any suitable method, for example a cellulose-based filter or a PES (polyethersulfone) filter. In a preferred embodiment, filtration of all solutions (before and after mixing the solutions) may be performed using a 0.22 micron filter with low affinity for proteins, such as a polyvinylidene fluoride (PVDF) filter. The molecular weight limit of the membrane of the filter is preferably about 5kDa or about 3 kDa.

Advantageously, the pharmaceutical composition of the invention is stable for at least 1 month, 3 months, 6 months, 9 months, 1 year or up to 2 years.

In a further aspect, the invention includes the use of arginine as a non-aggregating agent in a liquid pharmaceutical composition comprising IGF-1 and GH, preferably hgf-1 and hGH, more preferably rhIGF-1 and rhGH, wherein the concentration of arginine ranges from about 80mM to about 200mM, i.e., e.g., about 80mM, about 90mM, about 100mM, about 110mM, about 120mM, about 130mM, about 140mM, about 150mM, about 160mM, about 170mM, about 180mM, about 190mM or 200 mM.

It has been found that inclusion of an amino acid in a pharmaceutical composition enables a mixture of IGF-1 and GH to be formulated together as a clear solution formulation without loss of visual clarity of the mixture during subsequent refrigeration at 2 to 8 ℃ for at least 30 days, preferably at least 6 months, more preferably at least 12 months.

In a preferred embodiment of the invention, the formulation is stable for at least 18 months when stored at-20 ℃ or at a temperature of from 2 ℃ to 8 ℃.

In one embodiment, the present invention comprises a stable co-miscible formulation of the active ingredients human insulin-like growth factor 1(hIGF-1) and human growth hormone (hGH). In a preferred embodiment, the active ingredients are produced by recombinant methods and are designated rhIGF-1 and rhGH.

The formulation comprises rhIGF-1 and rhGH, a non-aggregating agent and a buffer. The formulation may comprise a surfactant, preferably a non-ionic surfactant, optionally a preservative and optionally a tonicity modifier and/or a bulking agent.

Preferably, the amino acid that enables the mixture of IGF-1 and GH to be formulated together into a clear solution formulation is arginine or lysine, more preferably arginine (e.g., arginine ion).

Preferably, the amino acid used as a non-aggregating agent is added separately to each solution before being mixed together in a clear solution formulation. More preferably, the final concentration of the non-aggregating agent in the clear solution is in the range of about 80mM to about 200mM, or in the range of about 100mM to about 180mM, or in the range of about 120mM to about 160mM, or in the concentration of about 150 mM.

The pH is adjusted to a value in the range of about 5 to about 7, preferably about 5.5 to about 6.5, more preferably about 5.8 to 6.2. In the context of pH, the term "about" means that the pH may vary by ± 0.2 or ± 0.1. The pH of the solution can be adjusted by any suitable method, for example, by adding an appropriate amount of an acidic solution, such as citrate, or preferably HCl.

According to the invention, the pH of the application may be, for example, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably 5.8, 6.2 or about 6.5.

In further embodiments, the osmotic modifier or tonicity modifier may be an inorganic salt. If included, the inorganic salt may be, for example, sodium chloride or potassium chloride, preferably sodium chloride, present in the composition at a concentration of 0 to 150mM, preferably at a concentration of 1 to 50 mM.

Furthermore, the optional preservative may be selected from the following list: phenol, benzyl alcohol, m-cresol and chlorobutanol. Preferred preservatives are phenol or benzyl alcohol. The preservative may be present in the composition at a concentration of about 0.1 to 5% (w/w), preferably about 0.2 to 2% (w/w), or more preferably about 1%.

The surfactants of the compositions disclosed in the present invention are for example selected from the following list: a polysorbate (tween) or a poloxamer, such as polysorbate 80, polysorbate 20 or poloxamer 188. Preferably, the surfactant is non-ionic, more preferably a polysorbate (tween), such as polysorbate 80, polysorbate 20, or a poloxamer, such as poloxamer 188, more preferably polysorbate 20 or poloxamer 188, at a concentration ranging from about 0.01 to 3% (w/w), preferably from about 0.03 to 0.50% (w/w), more preferably about 0.2% (w/w).

Furthermore, the buffer may be selected from a suitable pharmaceutically acceptable buffer having a pH of 5 to 6.5, such as sodium citrate or histidine or both, preferably acetate buffer, citrate buffer, phosphate buffer; amino acids such as histidine and all salts thereof, preferably the buffer is citrate or histidine. Preferably, the buffer is present in the final composition at a concentration of 1 to 100mM, preferably 1 to 50mM and most preferably about 10mM or about 20 mM.

According to the present invention, the amounts of IGF-1 and GH are about 2 to 40mg/mL (IGF-1) and about 1 to 12mg/mL (hGH), respectively, with preferred amounts being about 5 to 20mg/mL (IGF-1) and about 2 to 8mg/mL (hGH). Further preferred amounts are about 10mg/mL IGF-1 and about 3mg/mL hGH, or about 13.2mg/mL IGF-1 and 2mg/mL GH.

The weight ratio (w/w) of IGF-1: GH preferably ranges from 1:1 to 9:1, or alternatively from about 1:9 to 1: 1. More preferably, the weight ratio (w/w) of IGF-1: GH is selected from the following list: 9:1 (w/w); 6:1 (w/w); 3:1 (w/w); 2: 1; 3:7 (w/w); 1:1 (w/w); 1:2 (w/w); 1:5 (w/w); 7:3 (w/w); 9:1 (w/w).

More preferred weight ratios (w/w) of IGF-1: GH are selected from 1.1:1, 2.2:1, 3.3:1 and 6.6: 1. In one embodiment, the composition comprises a combination of rhIGF-1 and rhGH at concentrations of about 10 to 30mg/mL (IGF-1) and about 1 to 12mg/mL (rhGH), respectively, and in a weight ratio of IGF-1: GH of about 9:1 to 1:9(w/w), about 0.01 to 3% (w/w) of a surfactant, optionally about 0.1 to 5% (w/w) of a preservative, about 1 to 150mM of a buffer, preferably citrate or histidine, and a non-aggregating agent, such as arginine or lysine, at a concentration ranging from 80 to 200 mM. Optionally, the composition may further comprise one or two tonicity modifying agents, such as NaCl, KCl, at a concentration of NaCl and KCl of about 0 to 150mM and/or bulking agents, such as trehalose, mannitol, sorbitol or sucrose, at a concentration of 1 to 10% (w/w).

Furthermore, the present invention relates to a process for the preparation of a pharmaceutical composition comprising a combination of IGF-1 and GH.

In the pharmaceutical preparation of the present invention, human growth hormone and insulin-like growth factor are preferably produced by recombinant methods.

In a further embodiment, preferably in the compositions of the invention, IGF-1 and GH can be administered to a patient in each an effective amount or in each a sub-optimal but jointly effective amount. Preferably, such amounts are from about 25 to 250 micrograms IGF-1/kg body weight/day and from about 0.05-0.5mg GH/kg body weight/week.

Preferably, the pharmaceutical formulation is administered by injection, which is preferably parenteral, e.g. by subcutaneous, intramuscular, intravenous or infusion routes, the pharmaceutical composition will most preferably be used as a daily bolus injection, and preferably an Immediate Release Formulation (IRF).

The patient to be treated is preferably a mammal, in particular a human being, but it may also be an animal.

In a further embodiment, the invention provides the use of the composition for the preparation of a medicament for the treatment of a disease characterized by an increased or controlled amount of growth hormone in the plasma.

In particular, the present invention provides methods for treating growth hormone deficiency (GDH); turner's syndrome; prader-willi syndrome (PWS); methods and compositions for very low birth weight pediatric short stature (VLBW), adult GDH. Also such as endocrine disorders, for example, include administering to a patient suffering from a metabolic disorder characterized by a portion of endogenous growth hormone activity or signaling an amount of insulin-like growth factor-1 (IGF-1) and an amount of Growth Hormone (GH) that is effective in combination therapy to ameliorate a metabolic disorder in the patient. Wherein the patient has adult Idiopathic Short Stature (ISS) syndrome, wherein the patient receives a single administration of IGF-1 per day and receives a single administration of GH per day, and wherein the patient receives simultaneous administration of IGF-1 and GH.

The invention also provides methods and compositions for use in children with growth disorders characterized by partial endogenous growth hormone activity or signaling disorders. These growth causing childhood disorders continues to adulthood and affected adults may suffer from a variety of metabolic disorders.

According to the present invention, hGH and hIGF-1 are used as a medicament or pharmaceutical composition.

One valuable advantage of the present invention is to provide compositions that can be used as prefilled containers, such as syringes, or ready to use formulations.

The following examples serve to illustrate the invention without, however, limiting it.

Example 1

Solubility test

Prepared in a volume ratio range of 9:1 to 1:9(10mg/mL solution prepared in 50mM acetate buffer, pH 5.4) and(5mg/mL solution prepared in 10mM citrate buffer, pH 6). The mixture showed varying degrees of visible precipitation immediately or within hours of mixing.Andmass spectrometric analysis of the precipitate formed in the mixture showed the presence of two proteins in the precipitate. Table 1 summarizes the results for IGF-1, a commercially available productAnd GHObservations and results of clarity of the blends prepared.

TABLE 1

The solubility of IGF-1 was demonstrated to be greater than 20mg/mL throughout the pH range of the mixture (5.4-5.9), indicating that the solubility of IGF-1 was not responsible for the observed precipitate nor is IGF-1. The solubility of GH in citrate, acetate or histidine buffers in the pH range described has been found to be buffer dependent. The results indicate that the solubility of GH in acetate buffer solutions drops dramatically at pH values below 5.6, which may be responsible for the observed precipitate in the resulting mixture of solutions.

In comparison, however,andplacebo (without IGF-1, but other compositions andsame) or of a mixture ofAndplacebo (GH-free, but other composition with NutropinSame), indicating that the reduced solubility of the protein may also be associated with the interaction between the two proteins. In addition to this, the present invention is,by usingPlacebo diluted to a final concentration of 2.5mg/mL can be combined with NutropinThe IGF-1: hGH was mixed at a ratio of 2.2:1 or more without precipitation, indicating that the interaction between proteins was reversible.

Example 2

Comparison and preparation of blend compositions buffered in citrate at different pH

Lyophilized hGH was dissolved in 10mM citrate buffer pH6 containing 150mM sodium chloride and 0.2% polysorbate 20 to a final concentration of 5 mg/mL. The IGF-1 solutions in the different formulation buffers shown in column 1 of Table 1 were prepared by either dialysis of IGF-1 into the respective buffers or reconstitution of lyophilized IGF-1 in buffers. The final concentration of IGF-1 solution was 10mg/mL prior to mixing with the GH solution. The GH solution and IGF-1 solution were mixed together in various ratios as shown in table 2.

The visual appearance of the blends prepared from GH in citrate buffer and IGF-1 in various buffers at pH5.4 and 6 is summarized in table 2.

TABLE 2

The observations reported in table 2 indicate that precipitates were produced when IGF-1 solutions in various buffers were mixed with GH formulated in citrate buffer at pH 6.

Example 3

Preparation of the composition and clarity testing of the citrate buffered composition

Approximately 19mg/mL solutions of each protein (IGF-1 and hGH) were dialyzed separately into 10mM citrate buffer, pH 6.0, containing 10mM arginine. After overnight dialysis, the concentration of the solution was determined by measuring the Ultraviolet (UV) absorption at 280 nm. The final concentrations of IGF-1 solution and hGH solution were 14 and 21mg/mL, respectively. Each aliquot of each solution was constructed with the remaining excipients shown in Table 3 and diluted to a final protein concentration of 10 mg/mL. Each pair of separately formulated protein solutions (IGF-1 and hGH) was mixed at a 1:1 ratio to prepare a mixture containing 5mg/mL of each protein. After preparing the two protein mixtures, one of the two surfactants was added to each protein formulation and blend to give the final concentration. After 72 hours of refrigeration, the solution was examined. The two mixtures, which remained clear at this time (formulation compositions labeled a2 and a10 in table 3), were stored in a refrigerator and checked again to demonstrate that they remained clear after 70 days of storage. The results of the appearance test of the citrate formulation after 72 hours at 5 ℃ are summarized in table 3.

TABLE 3

Both clear formulations (A2 and A10) contained 100mM added arginine ion with little or no added sodium chloride. The compositions of the two formulations identified as a1 and a9 in table 3 were nearly identical to a2 and a10, respectively, but contained a smaller amount of added arginine ion (10mM), failing to remain clear.

Example 4

Preparation of histidine buffered compositions and comparative clarity test

A19 mg/mL solution of IGF-1 was dialyzed into 10mM histidine buffer, pH 5.6, containing 10mM arginine. After dialysis, the concentration of the solution was determined to be 18mg/mL by measuring the Ultraviolet (UV) absorption at 280 nm. The solution was constructed with appropriate amounts of additional arginine, benzyl alcohol, surfactant (polysorbate 20 or poloxamer 188), sodium chloride and mannitol to prepare the formulation compositions labeled B1 through B8 in table 4. Aliquots of the IGF-1-only formulation were used to reconstitute lyophilized growth hormone to prepare corresponding formulations containing 5mg/mL of each protein. The appearance of the solution was observed after refrigeration at 5 ℃ for 24 hours. In addition to the three formulations labeled B3, B4, and B8 in table 4, some precipitate appeared in all formulations at this time. These formulations were stored in the refrigerator for an additional 65 days and remained clear at the end of this time. The histidine buffered pH 5.6 formulations after 24 hours at 5 ℃ are summarized in Table 4.

TABLE 4

All three formulation mixtures that remained clear contained 100mM arginine ion. When observed after 24 hours of refrigeration, the formulation mixture labeled B7, which was just equivalent to the composition of the B8 formulation in table 4 but without the addition of arginine, appeared to precipitate, while the B8 formulation remained clear. Similarly, the formulation mixture labeled B4 in table 4 remained clear after long term refrigeration, whereas the B2 formulation containing only 10mM arginine and added sodium chloride failed to remain clear.

Example 5

Preparation of histidine buffered pH6 compositions

Approximately 19mg/mL solutions of each protein (IGF-1 and hGH) were dialyzed separately into 10mM histidine buffer, pH 6.0, containing 10mM arginine. After overnight dialysis, the concentration of the solution was determined by measuring the Ultraviolet (UV) absorption at 280 nm. The final concentrations of IGF-1 solution and hGH solution after dialysis were 11 and 21mg/mL, respectively. The solutions were separately constructed with appropriate amounts of additional arginine, benzyl alcohol, surfactant (polysorbate 20 or poloxamer 188), sodium chloride and mannitol to prepare two formulation compositions labeled C1 and C2 in table 5. Each protein formulation was mixed at a 1:1 ratio to prepare a blend and the appearance of all 6 solutions was observed after refrigeration at 5 ℃ for 72 hours. Both growth hormone formulations and blends showed some precipitation, probably due to the high salt concentration (150mM) in these formulations. Histidine buffer formulations at pH 6.0 after 72 hours at 5 ℃ are summarized in table 5.

TABLE 5

Example 6

Preparation and ratio of citrate and histidine buffered compositionsCompared with

Two formulations were prepared with each protein (IGF-1 and GH) at final protein concentrations of 20mg/mL (IGF-1) and 6mg/mL (GH), respectively:

preparation 1: 10mM citrate, 0.2% polysorbate 20, 1% benzyl alcohol, 100mM arginine, 50mM NaCl, pH 6.2

Preparation 2: 10mM histidine, 100mM arginine, 0.3% poloxamer 188, 1% benzyl alcohol, 50mM NaCl, pH 5.8

The formulation was prepared by buffer exchange of each protein into each of two buffers (buffer 1 and buffer 2) by tangential flow filtration to prepare four stock solutions of the concentrations shown in table 6. The preparation of the formulation stock solutions is summarized in table 6.

TABLE 6

Additional buffer and surfactant stock solutions were added to each stock solution with gentle mixing, followed by the addition of the appropriate amount of pure BzOH (benzyl alcohol to give the final composition of formulations 1 and 2 containing each protein). The protein concentrations of the IGF-1 and hGH formulations were 20mg/mL and 6mg/mL, respectively, and then the four formulations (two IGF-1 formulations and two hGH formulations) were diluted and/or mixed to give the final formulations and blends in Table 7. The solution was then stored at 2-8 ℃ until further dilution/bottling. All protein solutions were filter sterilized using PES membrane and then aliquoted into 3mL glass vials. The vials were stoppered, press sealed and stored in the refrigerator for up to 8 weeks. The appearance of each solution was evaluated at 2 week intervals over 8 weeks. At the end of 8 weeks, all 14 solutions were still clear and colorless. The results of the visual appearance of the citrate and histidine formulations containing 100mM arginine are summarized in table 7.

TABLE 7

The chemical stability of the compositions over 8 weeks was verified by periodic analysis of the formulations and blend. They were stored refrigerated at 5 ℃ and 25 ℃ to detect the initial stability-limited degradation products of IGF-1(des-Gly, Pro-IGF-1) and GH (deamidated GH), and to characterize the degradation rate and long-term stability of the registrations(IGF-1, injectable solution) anddegradation rates of (GH, injection) controls were compared. The degradation rates are shown in tables 8 and 9. The formulations showed no tendency for IGF-1 to show slow degradation over a period of 8 weeks at 5 ℃, however, storage of the new formulations and blend at 25 ℃ showed no change from that observedTypical accelerated degradation rates of (a) have comparable stability. Deamidation of GH in novel formulations and blends was shown at 5 ℃ and 25 ℃ with GHThe control had a comparable rate.

TABLE 8

^*Average degradation Rate of 15 batches

TABLE 9

Example 7

Preparation and comparison of histidine buffered IGF-1 compositions

IGF-1 was used to prepare two formulations with a final protein concentration of 20mg/mL (IGF-1):

preparation 1: 20mM histidine, 0.2% polysorbate 20, 1% benzyl alcohol, 150mM arginine, pH 5.8

Preparation 2: 50mM histidine, 0.2% polysorbate 20, 1% benzyl alcohol, 150mM arginine, pH 5.8

The preparation is prepared by exchanging proteins with each of two buffers via the buffers, adding a surfactant and a preservative, and mixing withStability was evaluated against the control. The stability data at 5 ℃, 25 ℃ and 40 ℃ are given in table 10.

Watch 10

Example 8

Stability data for Combo formulations

TABLE 11

¹Since the samples were stored at 5 ℃ until testing, the test data was used for the data at 5 ℃ and the date was taken for the most accurate accelerated stability data.

NT: the sample was not tested

These data were generated by size exclusion chromatography and provided stability for 1, 3 and 6 months.

Example 9

Establishment of alternative rhIGF-1 and rhGH co-formulation method

1. Materials and methods

1.1Raw material

The following raw materials were used in the study and are described in table 12:

12-raw material

Material	Suppliers of goods
		rhGH	Genentech
rhIGF-1	Lonza
		Citric acid	Sigma
arginine-HCl	Merck
		Phenol and its preparation	Merck
Poloxamer 188	BASF
		Sucrose	Beghin Say
Sodium hydroxide	Merck

WFI	Cooper
		Vial 5mL of form VB lyo	Schott
Plug 13mm	West-CTSU
		Crimps alu 13mm	West

1.2Device

The following equipment was used in the study:

-an autoclave FEDEGARI,

bottle sterile Polyethylene (PE) Nalgene ref.2019,

the box BIOMAX PES ref PXB005a50 for diafiltration,

-a clean room, a laminar flow hood,

-a Cogent μ Scale MillIPORE tangential flow filtration device,

filter MILLEX (33mm) PES 0.22 μm (MILLIPORE),

filter MILLEX (33mm) PVDF 0.22 μm (millipore),

-a glass beaker,

-a graduated cylinder having a graduated scale and a graduated scale,

-a magnetic stirrer having a magnetic field generator,

-a micropipette P1000,

-an oven FEDEGARI,

-a vial observation device according to the pharmacopoeia,

pump FLEXICON PF6n ° 212118,

-50 mL of syringe PE,

tube Eppendorf 1.5mL,

test tubes Falcon 50mL and 15mL,

tygon tube ((1.6mm + special needle),

-a washing machine CORIMA.

2. Method and formulation component optimization

And (4) carrying out method and preparation optimization experimental research. The formulation components are described in table 13 below:

13-Components of prototype (prototype) preparation A3-c

Ratio of	2.2:1	2.2:1
			Name (R)	A3	A3-c
Method strategy	A	A
			rhIGF-1[mg/mL]	7.9	7.9
rhGH[mg/mL]	3.6	3.6
			pH	6.0	6.0
Filler [ mM]	Sucrose 200	Sucrose 140
			Arginine HCl [ mM ]]	150	150
Histidine [ mM ]]	-	-
			Citrate salt [ mM ]]	20	20
Succinate [ mM ]]	-	-
			Poloxamer 188[ mg/mL ]]	2	2
Polysorbate 20[ mg/mL]	-	-
			Benzyl alcohol [ mg/mL ]]	-	-
Phenol [ mg/mL ]]	3.7	3.7
			Antioxidant [ mM ]]	-	-

2.1rhIGF-1 Tangential Flow Filtration (TFF) method

The rhIGF-1TFF method was performed using the parameters described below:

-quantifying IGF-1 to a predetermined: 80mL rhIGF-1, 25mg/mL,

-a diafiltration concentration of 25mg/mL,

-exchange of buffer: 20mM citrate, 150mM arginine, pH 6.0,

-Pellicon XL cassette: regenerated cellulose membrane, 5kDa molecular weight cut-off,

-TMP 18-22atm，

the pump is set at 12% power,

-6diavolume，

final rhIGF-1 concentration 30 mg/mL.

2.2Protein formulations：

1. In this alternative process, all excipients are first mixed together and then the protein is added to the excipients.

2. Excipient solutions were prepared at a pH below 6.0 so that addition of GH DS (pH about 7.5-8) would eventually result in a solution with a pH below 7.

2.2.1IGF-1 formulations

1. 8.44% of the final volume of citrate 80 mM/arginine 600, pH 5.5, was weighed into a glass beaker.

2. 3.7% of phenol of 10% final volume was added.

3. Add poloxamer 1882% in 10% final volume.

4. And (4) uniformly mixing.

5. 3.84g of sucrose was added.

6. Mixing, dissolving and homogenizing.

7. Measured pH value: 5.86.

8. while gently stirring the solution, a volume equivalent to 1.6g of diafiltered rhIGF-1 was added. The volume is calculated as follows:

9.52.7(mL) (volume of diafiltered IGF-1) =1600[ mg ] (amount of desired rhIGF-1)/30.36 [ mg/mL ] (IGF-1 concentration).

10. And (4) uniformly mixing.

11. Measured pH value: 6.05, so no pH adjustment is required.

12. WFI was added to give a final volume of 80 mL.

13. Measured pH value: 6.05.

14. filtration was done with a 0.22 μm PES filter.

15. Filtration was performed using a 0.22 μm PVDF filter.

2.2.2GH formulations

1. 25% of the final volume of citrate 80 mM/arginine 600, pH 5.5, was weighed into a glass beaker.

2. 3.7% of phenol of 10% final volume was added.

3. Add poloxamer 1882% in 10% final volume.

4. Add 10% final volume of WFI.

5. And (4) uniformly mixing.

a. Clear and very small amount of particles (possibly due to environmental reasons),

b. measured pH value: 5.9.

6. 4.83g of sucrose was added.

7. Mixing, dissolving and homogenizing.

a.pH：5.87。

8. 30mL GH DS was added while the solution was gently stirred.

9. And (4) uniformly mixing.

a. Measured pH value: 6.6.

b. final pH: 6.0.

10. filtration was done with a 0.22 μm PES filter.

11. Filtration was performed using a 0.22 μm PVDF filter.

2.2.3 Co-formulation (or blend)

Preparation of 45mL of the final blend rhIGF-1/rhGH (2.2: 1):

1. 17.9mL of IGF-1 formulation was added to 27.1mL of rhGH with magnetic stirring.

2. And (4) uniformly mixing.

3. The solution appeared clear and no visible particles.

4. Filtration with 0.22 μm PES filter in the clean room of building 2: a clear solution was obtained with no visible particles/easy to perform by syringe.

3. Use of alternative methods for selected formulations using histidine as a buffer

3.1Feasibility of preparation

rhIGF-1 formulations were formulated as § 2.2.1, rhGH formulations were formulated as § 2.2.2, and co-formulations 2.2:1 blends were formulated as § 2.2.3, except that:

histidine instead of citrate as a buffer,

-pH 6.0 instead of 5.8,

-polysorbate-20 as a surfactant instead of poloxamer-188,

pure benzyl alcohol as preservative instead of a 10% phenol solution,

2.5% HCl solution instead of 2.5% citric acid solution to adjust the pH,

PVDF filters are used for filtering individual protein products and blend preparations,

-preparing coformulations in a ratio of 1.1:1 and 6.6:1 instead of a 2.2:1 coformulation mixture.

3.2 optimization of filtration-repeatability

The treatment was carried out as described in § 3.1 (in the laboratory, 21 ℃) except that the filtration step was carried out in the order described below.

A first clarifying filtration with PES 0.22 μm for 150mL rhGH preparations + a second sterile filtration with PVDF.

A first clarifying filtration of 150mL rhGH preparation with 0.22 μm PVDF + a second sterile filtration with PVDF.

The following observations were made:

the rhGH solution appeared slightly milky and free of precipitate before clarification filtration.

After two filtrations, the rhGH preparation appeared to be particle free.

Visual inspection analysis demonstrated that the application of PVDF filters had a positive effect on the final sterile filtration of the formulation and co-formulation, i.e. a significant reduction of visible particles, and good stability when this type of filter was used.

Table 14:

formulations and co-formulation compositions

Example 10

Preparation method of rhIGF-1 and rhGH co-preparation

The rhGH substance (drug substance or DS) was a 20mg/mL solution in bicarbonate buffer at a concentration of 7.5 mM. rhIGF-1 substance (DS) was a 25-35mg/mL solution in 200mM citrate buffer.

The 3 different types of processes were prepared as follows (hereinafter referred to as process I, alternative processes a and B):

method I comprises the following steps:

rhGH and rhIGF-1 diafiltration for buffer exchange (original DS buffer vs histidine (His)20 mM/arginine (Arg)150mM pH 5.8).

-for each individual protein, formulating in the order of introduction described below:

addition of exchange buffer to adjust the DS concentration,

adding a polysorbate PS 20 solution into the mixture,

adding a Benzyl Alcohol (BA) solution,

-co-mixing the separate formulations to obtain a stable co-formulation.

The method A comprises the following steps:

rhGH and rhIGF-1 diafiltration for buffer exchange (original DS buffer vs histidine (His)20 mM/arginine (Arg)150mM pH 5.8).

-final formulation of each protein in the order of introduction described below:

the concentration of the buffer solution is carried out,

a solution of a surfactant in a solvent, in which,

the solution of the preservative(s) is,

WFI (water for injection),

the filler (if any),

the drug substance to be percolated through the membrane,

adjusting the pH with citric acid (or HCl)

WFI to final volume.

-co-mixing the separate formulations to obtain a stable co-formulation.

The method B comprises the following steps:

rhIGF-1 diafiltration for buffer exchange (raw DS buffer vs histidine (His)20 mM/arginine (Arg)150mM pH 5.8).

The undiluted rhGH substance, but a direct formulation of rhGH substance (i.e. rhGH DS was added to the excipient mixture without buffer exchange).

The final formulation is carried out in the same order as the order of introduction of the components in method a.

-co-mixing the separate formulations to obtain a stable co-formulation.

Claims (24)

1. A pharmaceutical composition comprising IGF-1 and GH in combination with

A non-aggregating agent;

a buffer;

a surfactant;

a preservative; and

a tonicity modifier which is a mixture of two or more,

wherein the non-aggregating agent is arginine present in the composition at a concentration ranging from 80mM to 200mM,

an IGF-1: GH weight ratio (w/w) in the range of 1:1 to 9:1,

the buffer is selected from histidine or citrate, at a concentration ranging from 1 to 50mM,

and the tonicity modifier is sodium chloride at a concentration of 1 to 50 mM.

2. The pharmaceutical composition of claim 1, wherein the non-aggregating agent concentration ranges from 100mM to 150 mM.

3. The pharmaceutical composition of claim 1, wherein the buffer has a concentration ranging from 10 to 20 mM.

4. The pharmaceutical composition of claim 1, wherein the surfactant is a non-ionic surfactant.

5. The pharmaceutical composition of claim 4, wherein the non-ionic surfactant is polysorbate 20 at a concentration ranging from 0.1 to 0.3% (w/w).

6. The pharmaceutical composition of claim 5, wherein the non-ionic surfactant is polysorbate 20 at a concentration of 0.2% (w/w).

7. The pharmaceutical composition of claim 4, wherein the non-ionic surfactant is poloxamer 188, at a concentration ranging from 0.1 to 0.5% (w/w).

8. The pharmaceutical composition of claim 7, wherein the non-ionic surfactant is poloxamer 188, at a concentration of 0.3% (w/w).

9. The pharmaceutical composition of claim 1, wherein the preservative is benzyl alcohol or phenol.

10. The pharmaceutical composition of claim 9, wherein the preservative is benzyl alcohol at a concentration ranging from 0.2 to 2% (w/w).

11. The pharmaceutical composition of claim 10, wherein the preservative is benzyl alcohol at a concentration of 1% (w/w).

12. The pharmaceutical composition of claim 1, wherein the IGF-1: GH weight ratio (w/w) ranges from 1:1(w/w) to 7:1 (w/w).

13. The pharmaceutical composition of claim 12, wherein the IGF-1: GH weight ratio (w/w) ranges from a value of 1.1:1(w/w), 2.2:1(w/w), 3.3:1(w/w), or 6.6:1 (w/w).

14. The pharmaceutical composition of claim 1, wherein the pH of the pharmaceutical composition ranges from 5.0 to 6.5.

15. The pharmaceutical composition of claim 14, wherein the pH of the pharmaceutical composition ranges from 5.4 to 6.2.

16. The pharmaceutical composition of claim 14, wherein the pH of the pharmaceutical composition ranges from 5.8 to 6.2.

17. The pharmaceutical composition according to claim 1, which is a ready-to-use formulation in a pre-filled syringe or in a syringe used in a syringe device.

18. A process for preparing a pharmaceutical composition according to claim 1, which process comprises:

a) preparing a hGH solution in a buffer comprising a non-aggregating agent and a tonicity modifying agent at pH5 to 6.5;

b) preparing an IGF-1 solution by dialyzing the IGF-1 preparation into the buffer used in step (a) comprising the non-aggregating agent and the tonicity modifying agent;

c) adding a surfactant and a preservative to the two stock solutions; and

d) the hGH solution and IGF-1 solution were mixed together.

19. A process for preparing a pharmaceutical composition according to claim 1, which process comprises:

a) preparing a solution I having or adjusted to a pH of 5.8 by mixing a buffer, a non-aggregating agent, a surfactant, a preservative, and optionally adjusting the volume with water;

b) preparing an IGF-1 solution in the buffer and non-aggregating agent used in step a) to obtain a solution II;

c) adding the solution II into the solution I to obtain a solution III;

d) preparing a solution IV having or adjusted to a pH of 5.8 by mixing buffer, non-aggregating agent, surfactant, preservative, and optionally adjusting the volume with water;

e) preparing a solution of GH, optionally comprising a sodium bicarbonate buffer, in the buffer and non-aggregating agent used in step d), to obtain solution V;

f) adding the solution V into the solution IV to obtain a solution VI;

g) optionally, filtering solutions III and VI independently;

h) mixing the filtered solutions III and VI in the ratio of IGF-1: GH (w/w) from 1:1 to 7:1(w/w) to obtain a solution VII; and

i) optionally, the solution VII is filtered.

20. The method according to claim 19, wherein the buffer is histidine, the non-aggregating agent is arginine, the surfactant is polysorbate 20, and the preservative is benzyl alcohol.

21. The method of claim 19, wherein the liquid GH drug substance is directly mixed with solution IV without performing step (e).

22. The method of claim 19, wherein the GH drug substance comprises sodium bicarbonate buffer.

23. The method of claim 19, wherein the filtering step is performed on a PVDF (polyvinylidene fluoride) filter.

24. The method of claim 19, wherein the non-aggregating agent is arginine at a concentration ranging from 100mM to 150 mM.