Stable liquid gonadotropin formulation

Abstract

The present invention pertains in general to the field of the stabilization of gonadotropin formulations, in particular liquid formulations of gonadotropins. The stabilization is achieved by a particular combination of excipients, preferably arginine and methionine. In a preferred embodiment, the formulation does not comprise a buffer.

Claims

1. A liquid pharmaceutical formulation comprising a gonadotropin, 50-160 mM arginine, and 0.05-1.5 mg/mL methionine, wherein the formulation does not additionally comprise a buffer, and wherein the pH of the formulation is between 6.0 and 7.5.

2. The pharmaceutical formulation of claim 1, wherein the gonadotropin comprises hCG (human chorionic gonadotropin), and optionally FSH and/or LH.

3. The pharmaceutical formulation of claim 1, wherein the gonadotropin comprises hMG (human menopausal gonadotropin).

4. The pharmaceutical formulation of claim 3, wherein the hMG is present in an amount of 300-900 IU/mL.

5. The pharmaceutical formulation of claim 3, wherein the hMG is present in an amount of 500-700 IU/mL.

6. The pharmaceutical formulation of claim 3, wherein the hMG is present in an amount of 600-650 IU/mL.

7. The pharmaceutical formulation of claim 3, wherein the hMG is present in an amount of about 625 IU/mL.

8. The pharmaceutical formulation of claim 1, wherein the gonadotropin comprises human origin, urinary-derived FSH, LH and/or hCG.

9. The pharmaceutical formulation of claim 1, wherein the gonadotropin comprises recombinant FSH, LH and/or hCG.

10. The pharmaceutical formulation of claim 1, further comprising a preservative.

11. The pharmaceutical formulation of claim 10, wherein the preservative is phenol.

12. The pharmaceutical formulation of claim 11, wherein the phenol is present in an amount of 4.0-6.0 mg/mL.

13. The pharmaceutical formulation of claim 1, further comprising a surface active agent.

14. The pharmaceutical formulation of claim 13, wherein the surface active agent is polysorbate 20.

15. The pharmaceutical formulation of claim 14, wherein the polysorbate 20 is present in an amount of 0.001-0.05 mg/mL.

16. The pharmaceutical formulation of claim 1, wherein the arginine is present in an amount of 80-160 mM.

17. The pharmaceutical formulation of claim 1, wherein the arginine is present in an amount of about 150 mM.

18. The pharmaceutical formulation of claim 1, wherein the arginine is L-arginine HCl.

19. The pharmaceutical formulation of claim 1, wherein the methionine is present in an amount of about 0.15 mg/mL methionine.

20. The pharmaceutical formulation of claim 1, wherein the pH of the formulation is 6.5-7.4.

21. The pharmaceutical formulation of claim 1, wherein the pH of the formulation is 6.5-7.1.

22. A method for treating infertility in a subject in need thereof, the method comprising administering the liquid pharmaceutical formulation of claim 1 to the subject.

Description

LIST OF FIGURES

(1) FIG. 1: Titration curve. The slope of the curves shows the buffer capacity, e.g. a steep slope depicts a low buffer capacity.

(2) FIG. 2: Response optimizer based on 25° C. results. Vertical lines show the level of L-arginine and L-methionine and the responses in the FSH and hCG immunoassay as Y (in IU/ml). R.sup.2.sub.HCG: 92.15% and R.sup.2.sub.FSH: 67.31%.

(3) All studies were confirmed by the additionally conducted real-time data.

EXAMPLES

Example 1

1 Background and Introduction

(4) Several different drug products containing gonadotropin hormones derived from urine of postmenopausal or pregnant women are used in the treatment of infertility, such as hMG (human menopausal gonadotropin) preparations. hMG possess Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) activity in a one to one ratio.

(5) FSH, LH and human chorionic gonadotropin (hCG) belong to the gonadotropins family of complex glycoprotein hormones. They are heterodimers composed of an α- and a β-subunit. The 92 amino acid α-subunit is common for these three gonadotropins, the β-subunits are unique, giving them their different biological characteristics (Wolfenson C. et al. 2005, Batch-to-batch consistency of human-derived gonadotropin preparations compared with recombinant preparations. Reproductive BioMedicine. Vol 10 No. 4:442-454; Shen, S. T., Cheng, Y. S., Shen, T. Y., and Yu, J. Y. 2006, Molecular cloning of follicle-stimulating hormone (FSH)-beta subunit cDNA from duck pituitary. Gen. Comp Endocrinol. 148:388-394; Fox, K. M., Dias, J. A., and Van, R. P. 2001, Three-dimensional structure of human follicle-stimulating hormone. Mol. Endocrinol. 15:378-389; Burova, T., Lecompte, F., Galet, C., Monsallier, F., Delpech, S., Haertle, T., and Combarnous, Y. 2001, Conformational stability and in vitro bioactivity of porcine luteinizing hormone. Mol. Cell Endocrinol. 176:129-134. The glycoprotein hormones all lose their bioactivity upon dissociation of the non-covalently linked subunits (Alevizaki, M. and Huhtaniemi, I. 2002, Structure-function relationships of glycoprotein hormones; lessons from mutations and polymorphisms of the thyrotrophin and gonadotropin subunit genes. Hormones. (Athens.) 1:224-232).

(6) LH and hCG binds to the same receptor and therefore both possess LH activity. In hMG the LH activity is originating mostly from hCG.

(7) It is the goal of the present invention to develop a gonadotropin formulation as a liquid formulation, for subcutaneous injection. For multidose injection, addition of a preservative typically is necessary (Meyer, B. K., Ni, A., Hu, B., and Shi, L. 2007, Antimicrobial preservative use in parenteral products: past and present. J. Pharm. Sci. 96:3155-3167; Chang, B. S. and Hershenson, S. Practical Approaches to Protein Formulation Development. In Rational Design of Stable Protein Formulations. J. F. Carpenter and M. C. Manning, editors. 2002, Plenum Publ., New York. 1-25; Pharmaceutical Formulation Development of Peptides and Proteins. 2000, CRC Press, Boca Raton).

(8) As in general, the native (bioactive) structure of proteins is very sensitive towards its surroundings e.g. the composition of the formulation, the container/closure systems, pH and temperature it is a difficult feat to identify a suitable liquid formulation for gonadotropins, different excipients were screened. In the present work the buffer capacity and real time stability of various hMG formulations are investigated using an FSH immunoassay, an hCG immunoassay, a bioassay, RP-HPLC for determination of oxidized proteins, titration and pH as described below in detail.

2 Product to be Studied

(9) 2.1 Materials

(10) 2.1.1 Drug Substance (DS)

(11) The hMG-HP drug substance (DS) was manufactured by Instituto Massone S. A. Argentina hMG-HP DS is received as a powder by Ferring Pharmaceuticals A/S, Copenhagen, Denmark.

(12) Determination of FSH and LH biological activity in the drug substance was performed according to the current version of the British Pharmacopeia (BP). It is also possible, if desired, to carry out this determination according to the USP 35 version. The FSH:LH bioactivity in hMG is 1:1 and therefore the average determined bioactivity of FSH and LH is used for compounding to drug product. Hence, when the concentration of hMG is given as e.g. 625 IU/ml it equals a bioactivity of 625 IU/ml FSH and 625 IU/ml LH.

(13) 2.1.2 Excipients

(14) A list of the excipients used in this work is described in Table 1.

(15) TABLE-US-00005 TABLE 1 List of excipients Name Quality Manufacturer Sodium hydroxide pellets Ph Eur, BP, JP, NF Merck Hydrochloric acid, Ph Eur, BP, JP, NF Merck fuming 37% Citric acid Monohydrate Ph Eur, BP, JPE, USP Merck ortho-Phosphoric acid 85% Ph Eur, BP, JPE, NF Merck Tri-sodium citrate dihydrate Ph Eur, BP, JPE, USP Merck Di-sodium hydrogen Ph Eur, BP, JPE, USP Merck phosphate dodecahydrate L-histidine EMPROVE ® exp Merck Ph Eur, USP L-methionine USP, multicompendial J.T. Baker L-arginine Ph Eur, USP Merck L-arginine Pharma Grade, EP, JP, Sigma-Aldrich monohydrochloride USP Sucrose EMPROVE ® exp Ph. Merck Eur, BP, JPE, NF D-(+)-trehalose dehydrate ≥99%, cGMP Sigma-Aldrich Mannitol EMPROVE ® Ph Eur, Merck BP, USP, JPE Lactose monohydrate Ph Eur, BP, NF, JP Merck Glycine EMPROVE ® Ph. Eur., Merck BP, JPE, USP Sodium Chloride EMPROVE, Ph. Eur., Merck BP, USP Polysorbate 20 Ph. Eur, NF, JPE J.T. Baker Poloxamer 188 Suitable for bio. Pharm. Merck Production, Ph. Eur., NF Phenol Ph. Eur, JP, USP Merck Meta-cresol Ph. Eur./USP parenteral Hedinger grade Milli-Q water — Millipore
2.1.3 Container Closure System

(16) For the stability studies, the primary packing materials were glass vials with rubber stoppers and alu/plastic caps, or glass cartridges with rubber plungers and crimp caps.

3 Manufacturing Procedure

(17) 3.1 Compounding

(18) All formulations are manufactured at lab-scale.

(19) For compounding of drug product solution (DP), stock solutions of each excipient and drug substance (DS) are mixed sequentially. Before adding hMG and final dilution to volume, the pH of each formulation is adjusted, when necessary. Stock solutions of all excipients and hMG are prepared in Milli-Q water.

(20) 3.2 Sterile Filtration and Aseptic Filling

(21) The formulations for stability are sterile filtered with Millipore PVDF 0.22 μm filter. Sterile filtration is performed in a LAF bench using autoclaved materials.

(22) The filling is performed after filtration. The containers are filled with sample solution. All vials and cartridges are filled under aseptic like conditions in a LAF bench and immediately closed with rubber stoppers or crimp caps. Outside the LAF bench the filled vials are sealed with aluminium flip-off caps.

4 Storage Conditions

(23) 4.1 Storage Conditions

(24) Containers containing drug product formulations are stored at accelerated conditions for up to 3 months at 30±2° C./65±5% RH and/or for up to a minimum of 6 months at 25±2° C./60±5% RH. At each storage temperature, the containers are stored in vertical positions. Cartridges are stored horizontally. All containers are protected from light.

5 Analytical Methods

(25) The analytical methods used in the studies are described below.

(26) 5.1 Titration Procedure

(27) According to USP-NF as described previously, a solution is said to be buffered if it resists changes in the activity of an ion on the addition of substances that are expected to change the activity of that ion. Buffers are substances or combinations of substances that impart this resistance to a solution. Buffered solutions are systems in which the ion is in equilibrium with substances capable of removing or releasing the ion. Buffer capacity refers to the amount of material that may be added to a solution without causing a significant change in ion activity. It is defined as the ratio of acid or base added (in gram-equivalents/L) to the change in pH units. The capacity of a buffered solution is adjusted to the conditions of use, usually by adjustment of the concentrations of buffer substances. Buffer capacity is expressed commonly as the number of equivalents (Eq) of strong acid or base that causes one liter of the solution in question to undergo one unit change in pH giving the unit mEq/(liter×pH) which was used for determination of the buffer capacity in the present invention. This means that the buffer capacity is defined as number of moles (equivalents) of H.sup.+/OH.sup.− needed to change the pH of one liter buffer solution by one unit.

(28) The buffer capacity was determined using placebo (placebo refers here to formulations without active ingredient) formulation adjusted to target pH as starting point. 0.2 N NaOH/HCl was used to titrate pH up or down. pH was measured after each addition of 0.2 N NaOH/HCl and the volume of 0.2 N NaOH/HCl was documented. The amount of 0.2 N NaOH/HCl was plotted as X-values and pH as Y-values. Fitted linear regression was performed around pH 6.8 for hMG placebo (target pH) and around pH 6.5 for reference placebo (target pH). The buffer capacity can be calculated as μL 0.2 N HCl/NaOH to move the pH by 0.01 pH unit/L DP and as milliequivalents (mEq) acid or base/(liter×pH unit), as described above in the definition.

(29) 5.2 FSH and hCG Immunoassay

(30) The FSH and hCG immunoassay was determined by sandwich ELFA.

(31) 5.3 Oxidized Proteins

(32) Oxidization of proteins was determined by RP-HPLC.

(33) 5.4 pH

(34) pH was measured according to Ph. Eur.

6 Results

(35) The results of the stability studies, the evaluation of the buffering agent in a buffer capacity study, as well as the stability results from the Design of Experiment (DoE) study are presented below.

(36) TABLE-US-00006 TABLE 2 Composition of liquid hMG (600 IU/ml) formulations Batch Preser- Stabilizer/ no. Buffer Surfactant vative Tonicity agent E-01 10 mM Phosphate.sup.1 0.005 mg/ml 3.0 mg/ml 41.3 mg/ml pH 6.8 polysorbate 20 M-cresol Mannitol E-02 10 mM Phosphate.sup.1 0.005 mg/ml 3.0 mg/ml 85.9 mg/ml pH 6.8 polysorbate 20 M-cresol Trehalose E-03 10 mM Phosphate.sup.1 0.1 mg/ml 3.0 mg/ml 74.3 mg/ml pH 6.8 Poloxamer 188 M-cresol Sucrose E-04 10 mM Phosphate.sup.1 0.1 mg/ml 3.0 mg/ml 18.8 mg/ml pH 6.8 Poloxamer 188 M-cresol Glycine E-05 10 mM Phosphate.sup.1 0.005 mg/ml 5.0 mg/ml 67.7 mg/ml pH 6.8 polysorbate 20 Phenol Sucrose E-06 10 mM Phosphate.sup.1 0.005 mg/ml 5.0 mg/ml 6.8 mg/ml pH 6.8 polysorbate 20 Phenol NaCl E-07 10 mM Phosphate.sup.1 0.1 mg/ml 5.0 mg/ml 37.6 mg/ml pH 6.8 Poloxamer 188 Phenol Mannitol E-08 10 mM Citrate.sup.2 0.005 mg/ml 3.0 mg/ml 71.1 mg/ml pH 6.8 polysorbate 20 M-cresol Sucrose E-09 10 mM Citrate.sup.2 0.005 mg/ml 3.0 mg/ml 29.3 mg/ml pH 6.8 polysorbate 20 M-cresol L-arginine HCl E-10 10 mM Citrate.sup.2 0.1 mg/ml 3.0 mg/ml 39.5 mg/ml pH 6.8 Poloxamer 188 M-cresol Mannitol E-11 10 mM Citrate.sup.2 0.005 mg/ml 5.0 mg/ml 74.7 mg/ml pH 6.8 polysorbate 20 Phenol Trehalose E-12 10 mM Citrate.sup.2 0.1 mg/ml 5.0 mg/ml 64.5 mg/ml pH 6.8 Poloxamer 188 Phenol Sucrose E-13 10 mM Citrate.sup.2 0.1 mg/ml 5.0 mg/ml 6.5 mg/ml pH 6.8 Poloxamer 188 Phenol NaCl E-14 10 mM Histidine 0.005 mg/ml 3.0 mg/ml 77.5 mg/ml pH 6.8 polysorbate 20 M-cresol Sucrose E-15 10 mM Histidine 0.1 mg/ml 3.0 mg/ml 19.6 mg/ml pH 6.8 Poloxamer 188 M-cresol Glycine .sup.110 mM Di-sodium hydrogen phosphate dodecahydrate, pH adjusted with phosphoric acid .sup.210 mM Tri-sodium citrate dihydrate, pH adjusted with citric acid
6.1 Stability of Liquid hMG Formulation/3 Months

(37) Protein molecules can be stabilized by adding excipients to the solution e.g. salts, carbohydrates or amino acids but the degree of stabilization upon addition of different carbohydrates, salts and amino acids varies massively between different formulations. Here, the initial stability studies were performed to screen various stabilizers in combination with preservatives and buffers in a liquid formulation of hMG. Surprisingly, the results show a superior stabilizing effect of L-arginine as shown in Tables 3 and 6 for the hCG immunoassay and Tables 4 and 7 for the FSH immunoassay. Formulation E-09 exhibits a superior stability compared to all other formulations. Formulation E-09 is the only formulation which contains L-arginine (see Table 2). Formulation K-01, K-03 and K-04 as well as K-09 exhibit superior stability compared to all other formulations (see Table 6). These formulations again contain L-arginine (see Table 5). In Table 4, formulation E-09 excels with best stability compared to all other formulations also for the FSH immunoassay and Table 7 confirms that formulation K-09 shows the highest FSH immunoassay result compared to all other formulations.

(38) TABLE-US-00007 TABLE 3 hCG immunoassay during 1 month storage at 30 ± 2° C./65 ± 5% RH. Formulation E-09 exhibits superior stability compared to all other formulations. Formulation E-09 contains L-arginine, see Table 2. hCG [% of initial] 30° C. ± 2° C./65 ± 5% RH Formulation Initial 0.5 month 1 month E-01 100 91 85 E-02 100 88 87 E-03 100 87 80 E-04 — — — E-05 100 83 81 E-06 100 101 100 E-07 100 89 87 E-08 100 89 81 E-09 (L-arginine HCl) 100 113 106 E-10 100 95 91 E-11 100 99 89 E-12 100 94 87 E-13 100 99 86 E-14 — — — E-15 — — —
(Formulation E-04, E-14 and E-15 excluded from testing due to coloration)

(39) TABLE-US-00008 TABLE 4 FSH immunoassay after 3 months storage at 30 ± 2° C./65 ± 5% RH. Formulation E-09 exhibits the best stability compared to all other formulations. Formulation E-09 contains L-arginine, see Table 2. FSH [% of initial] 30° C. ± 2° C./65 ± 5% RH Formulation Initial 3 months E-01 100 83 E-02 100 77 E-03 100 83 E-04 — — E-05 100 84 E-06 100 86 E-07 100 86 E-08 100 84 E-09 (L-arginine) 100 95 E-10 100 83 E-11 100 83 E-12 100 89 E-13 100 85 E-14 — — E-15 — —
(Formulation E-04, E-14 and E-15 excluded from testing due to coloration)
6.2 Stability of Liquid hMG Formulation—3 Months
From initial screening additional formulations with arginine was screened.

(40) TABLE-US-00009 TABLE 5 Composition stability study for liquid hMG 600 IU/ml-Overview of formulations Batch Stabilizer/ no. Buffer Surfactant Preservative Antioxidant Tonicity agent K-01.sup.1 10 mM Citrate.sup.5 0.005 mg/ml 3.0 mg/ml 1.5 mg/ml 28.0 mg/ml pH 6.8 Polysorbate 20 M-cresol L-methionine L-arginine HCl K-02  1 mM Phosphate.sup.4 0.005 mg/ml 3.0 mg/ml 1.0 mg/ml 21.0 mg/ml pH 6.8 Polysorbate 20 M-cresol L-methionine Lactose  7.0 mg/ml NaCl K-03 10 mM Citrate.sup.5  0.1 mg/ml 5.0 mg/ml 1.5 mg/ml 25.3 mg/ml pH 6.8 Poloxamer 188 Phenol L-methionine L-arginine HCl K-04 10 mM Phosphate.sup.4 0.005 mg/ml 3.0 mg/ml 1.5 mg/ml 29.3 mg/ml pH 6.8 Polysorbate 20 M-cresol L-methionine L-arginine HCl K-05.sup.2 10 mM Citrate.sup.5  0.1 mg/ml 5.0 mg/ml 1.0 mg/ml 67.4 mg/ml pH 6.8 Poloxamer 188 Phenol L-methionine Sucrose K-06 10 mM Citrate.sup.5  0.1 mg/ml 3.0 mg/ml 1.0 mg/ml 74.6 mg/ml pH 6.8 Poloxamer 188 M-cresol L-methionine Sucrose K-07.sup.3 10 mM Citrate.sup.5  0.1 mg/ml 3.0 mg/ml 1.0 mg/ml 38.3 mg/ml pH 6.8 Poloxamer 188 M-cresol L-methionine Mannitol K-08 10 mM Citrate.sup.5  0.1 mg/ml 5.0 mg/ml 1.0 mg/ml 34.7 mg/ml pH 6.8 Poloxamer 188 Phenol L-methionine Mannitol K-09.sup.6 10 mM Citrate.sup.5  0.1 mg/ml 3.0 mg/ml 1.5 mg/ml   20 mg/ml pH 6.8 Poloxamer 188 M-cresol L-methionine L-arginine HCl 22.1 mg/ml Sucrose .sup.1Same as formulation E-09 in table 2 .sup.2Same as formulation E-12 in table 2 .sup.3Same as formulation E-10 in table 2 .sup.410 mM Di-sodium hydrogen phosphate dodecahydrate, pH adjusted with phosphoric acid .sup.510 mM Tri-sodium citrate dihydrate, pH adjusted with citric acid .sup.6Strength is 530 IU/ml

(41) TABLE-US-00010 TABLE 6 hCG immunoassay during 3 months storage at 25 ± 2° C./ 60 ± 5% RH. Formulations K-01, K-03, K-04 and K-09 exhibit superior stability compared to all other formulations. Formulation K-01, K-03, K-04 and K-09 contain L-arginine, see Table 5 hCG [% of initial] 25° C. ± 2° C./65 ± 5% RH Formulation Initial 1 month 2 months 3 months K-01 (L-arginine) 100 110 105 105 K-02 100 100 68 57 K-03 (L-arginine) 100 110 106 104 K-04 (L-arginine) 100 109 106 113 K-05 100 94 82 71 K-06 100 98 76 80 K-07 100 85 74 67 K-08 100 93 79 71 K-09 (L-arginine) 100 99 101 100

(42) Very clearly, the formulations comprising L-arginine are much more stable than those comprising different stabilizing agents. Compare e.g. formulation K-02 and K-03 after three months.

(43) TABLE-US-00011 TABLE 7 FSH immunoassay during 3 months storage at 25 ± 2° C./ 60 ± 5% RH. Formulation K-09 show highest FSH immunoassay result compared to all other formulations. Formulation K-09 contains L-arginine, see Table 5 FSH [% of initial] 25° C. ± 2° C./60 ± 5% RH Formulation Initial 1 month 2 months 3 months K-01 (L-arginine) 100 112 112 111 K-02 100 104 106 87 K-03 (L-arginine) 100 108 120 107 K-04 (L-arginine) 100 108 112 112 K-05 100 107 116 111 K-06 100 109 117 115 K-07 100 101 112 104 K-08 100 107 107 109 K-09 (L-arginine) 100 112 120 119

(44) The results for hCG are also confirmed for FSH—compare e.g. formulation K-02 and K-03 after three months.

(45) 6.3 Buffer Capacity Study

(46) Buffering agents e.g. sodium phosphate and sodium citrate are physiologically tolerated buffers and are quite commonly added to maintain the pH in a desired range. Tri-sodium citrate dihydrate and Di-sodium hydrogen phosphate dodecahydrate are evaluated as buffering agents by means of buffer capacity. The buffer capacity is calculated as the amount of acid or base needed to move the pH a predefined step, i.e. the amount of μL 0.2 N HCl/NaOH needed to move the pH by 1 pH unit/L DP calculated as mEq acid or base/(liter×pH unit) as explained above.

(47) TABLE-US-00012 TABLE 8 Composition buffer capacity study-Overview Formulations Batch Stabilizer/ no. Buffer Surfactant Preservative Antioxidant Tonicity agent C-01 No buffer 0.005 mg/ml 5.0 mg/ml 1.5 mg/ml 120 mM L-arginine pH 6.8 Polysorbate 20 Phenol L-methionine HCl.sup.4 C-02  5 mM 0.005 mg/ml 5.0 mg/ml 1.5 mg/ml 120 mM L-arginine Citrate.sup.1 Polysorbate 20 Phenol L-methionine HCl.sup.4 pH 6.8 C-03 10 mM 0.005 mg/ml 5.0 mg/ml 1.5 mg/ml 120 mM L-arginine Citrate.sup.1 Polysorbate 20 Phenol L-methionine HCl.sup.4 pH 6.8 C-04.sup.3  1 mM.sup.2 0.005 mg/ml 5.0 mg/ml 1.0 mg/ml 31.76 mg/ml Phosphate Polysorbate 20 Phenol L-methionine Na.sub.2SO.sub.4 × 10 H.sub.2O pH 6.5 (14 mg/ml Sodium sulphate) .sup.1Tri-sodium citrate dihydrate .sup.20.8 mM Na.sub.2HPO.sub.4 × 12 H.sub.2O and approx. 0.2 mM H.sub.3PO.sub.4 to pH 6.5 .sup.3Reference placebo .sup.4Equal to 25.3 mg/ml L-arginine HCl

(48) The result of the titration conducted to determine buffer capacity is depicted in FIG. 1. The slope of the curves shows the buffer capacity, e.g. a steep slope represents a low buffer capacity. From FIG. 1 it can be observed that batch C-04 (reference formulation) has the lowest buffer capacity. In the relevant pH area 6.8±0.3 for hMG, all hMG formulations C-01 (no buffer), C-02 (5 mM Tri-sodium citrate dihydrate) and C-03 (10 mM Tri-sodium citrate dihydrate) have similar buffer capacity and a higher buffer capacity than the reference formulation containing 1 mM Di-sodium hydrogen phosphate dodecahydrate. The target pH for the reference formulation is 6.5. Formulation C-01, C-02 and -03 are compared with the reference which is known to have a stable pH. Numerous stability studies of the reference formulation C-04 have been conducted without any observed drifts in pH.

(49) The following Table 9 shows the results of the buffer capacity study:

(50) TABLE-US-00013 TABLE 9 Buffer capacity study. In the formulation relevant pH area 6.8 for hMG, all hMG formulations have a higher buffer capacity than the reference placebo formulation. Buffer capacity Buffer expressed as: capacity mEq acid or around a α base/(liter × Batch Buffer target pH pH range (slope) pH unit) C-01 No buffer 6.8 6.564-6.947 0.001507 1.32 C-01 No buffer 7.0 6.786-7.193 0.000822 2.44 C-02  5 mM 6.8 6.599-6.951 0.000672 2.96 Trisodium citrate dihydrate C-03 10 mM 6.8 6.572-6.946 0.000750 2.66 Trisodium citrate dihydrate C-04  1 mM 6.5 6.199-6.752 0.002700 0.74 (reference) Disodium hydrogen phosphate dodecahydrate C-04  1 mM 6.8 6.582-6.982 0.002091 0.96 (reference) Disodium hydrogen phosphate dodecahydrate
(C-01 and C-04 is mentioned twice because the buffer capacity was calculated in pH ranges around two different pH values respectively)

(51) Surprisingly, the buffer capacity of the hMG formulation with no buffer (C-01) is higher than the reference formulation C-04. The main excipient component in the hMG formulation is L-arginine at a concentration of 120 mM. The pKa values for L-arginine are pKa.sub.1=2.17; pKa.sub.2=9.04 and pKa.sub.3=12.5 (Handbook of Pharmaceutical Excipients. 2015, Pharmaceutical Press, London). It is very unexpected that arginine exhibits sufficient buffering behaviour far from the pKa values.

(52) 6.4 Oxidation Studies

(53) After 3 months of storage at 30±2° C./65±5% RH, formulation E-09 containing L-arginine (see Table 5) has increased the level of oxidized proteins by 241% of the initial value (see table 10 below). This is a very high amount and there are instances where such high amounts of oxidation are not desirable.

(54) Therefore, further studies were conducted to research whether this problem could be overcome.

(55) There are several choices of antioxidants that can be used in protein formulations. Methionine can be added to prevent oxidation, by a proposed mechanism of competing with oxidation of the methionine residues in the proteins. The result of adding methionine to a formulation containing arginine is shown in Table 10. Formulation K-01, K-03, K-04 and K-09 contain arginine and methionine and the level of oxidized proteins is clearly and advantageously reduced. Formulation K-01 (with methionine) is the same formulation as formulation E-09 (without methionine). Comparing these two batches it can be seen that the level of oxidized proteins is reduced tremendously by methionine.

(56) TABLE-US-00014 TABLE 10 Oxidized proteins % increase from initial during 3 months storage at 30 ± 2° C./65 ± 5% RH. Formulation E-09 contains arginine and no methionine. Formulation K-01, K-03, K-04 and K-09 contain arginine and methionine, see Table 5 Oxidized proteins [% 30° C. ± 2° C./ increase from initial] 65 ± 5% RH Formulation 3 months E-01 44 E-02 39 E-03 47 E-04 — E-05 37 E-06 43 E-07 36 E-08 51 E-09 (arginine and no methionine) 241 E-10 55 E-11 47 E-12 53 E-13 46 E-14 — E-15 — K-01 (arginine and methionine) 24 K-03 (arginine and methionine) 12 K-04 (arginine and methionine) 8 K-09 (arginine and methionine) 13
(Formulation E-04, E-14 and E-15 excluded due to coloration)

(57) In conclusion, these two studies demonstrate a tremendously stabilizing effect of arginine and an anti-oxidizing effect even of low amounts of methionine in a formulation of gonadotropins.

(58) 6.5 DoE Stability Study

(59) Combining the results from the above studies, and the buffer capacity study, a DoE study was performed to investigate the interactions between arginine and methionine and to determine the optimal concentrations of these two excipients.

(60) TABLE-US-00015 TABLE 11 Compositions for DoE stability study for liquid hMG 600 IU/ml-overview of formulations Batch Stabilizer/ no. Buffer.sup.2 Surfactant Preservative Antioxidant Isotonicity agent D-01 No buffer 0.005 mg/ml 5.0 mg/ml 1.5 mg/ml  80 mM pH 6.8 Polysorbate 20 Phenol L-methionine L-arginine HCl D-02 No buffer 0.005 mg/ml 5.0 mg/ml 1.5 mg/ml 160 mM pH 6.8 Polysorbate 20 Phenol L-methionine L-arginine HCl D-03 No buffer 0.005 mg/ml 5.0 mg/ml 0.8 mg/ml 120 mM pH 6.8 Polysorbate 20 Phenol L-methionine L-arginine HCl D-04 No buffer 0.005 mg/ml 5.0 mg/ml 0.8 mg/ml 120 mM pH 6.8 Polysorbate 20 Phenol L-methionine L-arginine HCl- D-05 No buffer 0.005 mg/ml 5.0 mg/ml 0.1 mg/ml 160 mM pH 6.8 Polysorbate 20 Phenol L-methionine L-arginine HCl D-06 No buffer 0.005 mg/ml 5.0 mg/ml 0.1 mg/ml  80 mM pH 6.8 Polysorbate 20 Phenol L-methionine L-arginine HCl D-07.sup.3 No buffer  0.1 mg/ml 5.0 mg/ml 1.5 mg/ 120 mM pH 6.8 Poloxamer 188 Phenol L-methionine L-arginine HCl D-08.sup.3 10 mM Citrate.sup.1  0.1 mg/ml 5.0 mg/ml 1.5 mg/ 120 mM pH 6.8 Poloxamer 188 Phenol L-Methionine L-arginine HCl .sup.1Tri-sodium citrate dehydrate 2pH is adjusted with 0.2N HCl/NaOH for formulations without buffer and 0.2N NaOH/0.5M .sup.3Citric acid Monohydrate for formulation with Tri-sodium citrate dihydrate buffer Included as reference formulations
6.5.1 FSH Immunoassay

(61) Stability results of FSH immunoassay during storage for 3 months at 25±2° C./60±5% RH are listed in Table 12.

(62) TABLE-US-00016 TABLE 12 Results of FSH immunoassay during storage at 25 ± 2° C./60 ± 5% RH. The full description of all formulations is listed in Table 11. FSH [IU/ml] 25° C. ± 2° C./60 ± 5% RH Formulation Initial 1 month 2 months 3 months D-01 516 501 468 462 D-02 521 492 503 478 D-03 511 503 513 474 D-04 515 505 490 477 D-05 532 521 513 473 D-06 518 488 458 493 D-07 518 528 482 490 D-08 514 499 472 481

(63) Statistical calculation was performed for FSH activity [IU/ml] to evaluate the influence and interaction of arginine and—methionine. The result of the statistical evaluation confirms that L-arginine has a statistical significant influence on FSH immunoassay stability results. Methionine has no statistical significant influence on FSH immunoassay stability results. There is no statistical significant interaction between arginine and methionine for this response parameter.

(64) 6.5.2 hCG Immunoassay

(65) Stability results of hCG immunoassay during storage for 3 months at 25±2° C./60±5% RH are listed in Table 13 Table.

(66) TABLE-US-00017 TABLE 13 Results of hCG immunoassay during storage at 25 ± 2° C./60 ± 5% RH. The full description of all formulations is listed in Table 11. hCG [IU/ml] 25° C. ± 2° C./60 ± 5% RH Formulation Initial 1 month 2 months 3 months D-01 83 82 75 56 D-02 87 83 93 76 D-03 93 93 88 70 D-04 86 91 87 70 D-05 90 98 92 85 D-06 87 85 72 61 D-07 93 92 88 71 D-08 91 96 94 81

(67) Statistical calculation was performed for hCG activity [IU/ml] to evaluate the influence and interaction of arginine and methionine. The result of the statistical evaluation confirms that arginine has a statistical significant influence on hCG immunoassay stability results. Methionine content has a minor influence at 25° C. on hCG immunoassay stability results. It becomes clear that over a low (0.1 mg/ml) concentration of methionine no further increase in stability by increasing the concentration even up to 1.5 mg/ml is detectable. There is no statistical significant interaction between arginine and methionine for this response parameter.

(68) 6.5.3 Oxidized Proteins

(69) In the above stability studies it was surprisingly observed that the presence of arginine has a tremendously stabilizing effect. However, addition of arginine resulted in an increased level of oxidized proteins. Methionine can be added to prevent this oxidation and this study investigates the concentration balance between arginine as stabilizer and methionine as antioxidant.

(70) Stability results of amount of oxidized proteins [% increase from initial] during storage for 6 months at 25±2° C./60±5% RH and 3 month at 30±2° C./65±5% RH for formulations with arginine and Methionine (in the concentration range 0.1-1.5 mg/ml) are listed below. For comparison formulation E-09 with arginine and without methionine is also included in Table 14 Table.

(71) TABLE-US-00018 TABLE 14 Results of oxidized proteins during storage at 25 ± 2° C./60 ± 5% RH and 30 ± 2° C./ 65 ± 5% RH. The full description of all formulations is given in Table 11. Oxidized proteins [% increase from initial] 30° C. ± 2° C./65 ± 5% RH 25° C. ± 2° C./60 ± 5% RH Formulation 1 month 2 months 3 months 1 month 2 months 3 months 6 months D-01 5.5 4.1 8.3 10.1 4.1 9.6 6.9 D-02 9.9 8.5 10.3 11.7 11.7 6.6 8.5 D-03 10.9 10.0 11.4 12.3 13.3 11.8 12.8 D-04 8.9 8.4 8.9 8.9 11.2 9.8 8.9 D-05 12.9 13.8 13.8 13.3 20.5 11.0 15.2 D-06 7.0 9.8 11.6 10.7 7.9 9.3 11.2 D-07 14.0 10.1 11.1 14.5 13.5 15.0 10.1 D-08 7.6 9.5 7.6 13.3 9.0 11.8 7.6 E-09 — — 241 — — — — (no L-methionine)

(72) From Table 14 Table it is surprisingly seen that even small quantities of methionine are enough to prevent oxidation in the entire concentration range of arginine.

(73) 6.5.4 Summary of DoE

(74) To conclude and summarize the results of the DoE, the response optimizer in Minitab was applied, see FIG. 2. The Minitab Response Optimizer tool shows how different experimental factor settings e.g. arginine and methionine concentrations affect the predicted responses e.g. the FSH immunoassay [IU/ml] and the hCG immunoassay [IU/ml] for the factorial design. The optimization plot shows the effect of each factor (columns) on the responses (rows). The vertical bold lines on the graph represent the current factor settings. The numbers displayed at the top of a column show the current factor level settings (in squared brackets). The horizontal dashed lines and corresponding numbers represent the responses for the current factor level. The response optimizer will be based on 25° C. results. The result of applying the response optimizer is depicted FIG. 2. The best stability (composite desirability) is obtained at high concentrations of arginine and low concentrations of methionine.

(75) 6.6 pH Study

(76) In the buffer capacity study it was shown that a buffering agent was not necessary in the hMG formulation, by using only arginine to stabilize the pH in the desired pH range.

(77) To confirm that the pH is maintained during storage, it was measured during 6 months of storage at 25±2° C./60±5% RH and 3 months at 30±2° C./65±5% RH. The results are listed in Table 15.

(78) Target pH for all formulations was pH 6.8 and pH of all formulations was 6.8 at the initial time point. pH was fairly stable but increased slightly to about 6.9 after 2 months and maintained a pH around 6.9 up to 6 months of storage. Results up to 6 months of storage confirm stability of pH in the tested concentration range of arginine.

(79) TABLE-US-00019 TABLE 15 Results of pH study 30° C. ± 2° C./ 25° C. ± 2° C./ 65 ± 5% RH 60 ± 5% RH pH 2 3 2 3 6 Formulation Initial month months months months months D-01 6.8 6.9 6.9 6.9 6.9 6.9 D-02 6.8 6.9 6.9 6.9 6.9 6.8 D-03 6.8 6.9 6.9 6.9 6.9 6.8 D-04 6.8 6.9 6.9 6.9 6.9 6.8 D-05 6.8 6.9 6.9 6.9 6.9 6.8 D-06 6.8 6.9 6.9 6.9 6.9 6.8 D-07 6.8 6.9 6.9 6.9 6.9 6.8 D-08 6.8 6.9 6.9 6.9 6.9 6.9

7 Conclusion

(80) Very surprisingly, the buffer capacity of the hMG formulation with no buffer is higher than the reference with 1 mM Di-sodium hydrogen phosphate dodecahydrate. The main excipient component in the hMG formulation is arginine. The pKa values for arginine are pKa.sub.1=2.17; pKa.sub.2=9.04 and pKa.sub.3=12.5. It is unexpected that arginine exhibits sufficient buffering behaviour far from its pKa values. The results however clearly show that no—additional buffering agent is needed in the hMG formulation in the current pH range. Arginine alone is sufficient to maintain the pH in the desired pH range. Stability of the pH was confirmed in the DoE study testing a concentration range of arginine during 6 months storage at 25±2° C./60±5% RH and 3 months storage at 30±2° C./65±5% RH which confirmed the observations in the buffer capacity study.

(81) Addition of arginine to the inventive liquid hMG formulation was shown to have a tremendously stabilizing effect on liquid hMG formulations. However, arginine was also found to result in a high level of oxidized proteins. Addition of methionine was shown to prevent oxidation compared to a formulation without methionine.

(82) During 6 months of storage at 25±2° C./60±5% RH and 3 months of storage at 30±2° C./65±5% RH, it was shown that even a small concentration of methionine prevents oxidation.

(83) Concluding the results from these studies it is clearly seen that the addition of arginine is sufficient to maintain pH at the desired pH level. Addition of arginine stabilizes the tested gonadotropin formulation tremendously in a liquid formulation. No other of the tested amino acid, sugar or salt showed a similar stabilizing effect. The level of oxidized proteins is increased significantly by addition of arginine, however addition of even low amounts of methionine prevents protein oxidation. Surprisingly, even small quantities of methionine are sufficient to prevent oxidation independently of the concentration of arginine.

Example 2

(84) The present inventors further confirmed that the present advantageous inventive formulation would also be suitable to stabilize the respective recombinant gonadotropin formulation.

(85) To that avail, recombinant FSH and recombinant hCG (with the sequences as described above, respectively) were prepared according to well known methods.

(86) An accelerated stability study was carried out for two formulations, comprising rhCG or rFSH, respectively, as described below.

(87) The observed surprising stabilising effect of arginine has also been confirmed in recombinant proteins. Both rFSH and rhCG have been formulated in 5 mg/ml phenol, 0.15 mg/ml L-methionine, 150 mM arginine HCl, 0.005 mg/ml polysorbate 20, pH 6.8. To simplify the analysis of the protein stability, rFSH and rhCG were formulated in different containers.

(88) TABLE-US-00020 TABLE 16 Immunoactivity (IU/ml] 1 month, 2 months, 4 months, 16 months, Initial 30° C. 30° C. 30° C. 5° C. FSH [33.3 μg/ml] 464 444 456 469 435 rhCG [50 μg/ml] 711 680 656 600 685 SEC (Purity of rFSH, %) 1 month, 2 months, 4 months, 16 months, Initial 30° C. 30° C. 30° C. 5° C. FSH [33.3 μg/ml] 98.9 98.6 98.3 97.9 96.9 HIC (Purity of rhCG, %) 1 month, 2 months, 4 months, 14 months, Initial 30° C. 30° C. 30° C. 5° C. rhCG [50 μg/ml] 98.8 96.8 93.3 89.4 99.9

(89) This fully confirms that the present inventive formulation with arginine, but no additional buffer, and with a low amount of methionine is suitable to stabilized recombinant gonadotropins as well.

Example 3

(90) Further studies were conducted, wherein the following composition was assessed:

(91) TABLE-US-00021 TABLE 17 Composition for liquid hMG 625 IU/ml formulation Stabilizer/ Batch Tonicity no. Buffer.sup.1 Surfactant Preservative Antioxidant agent J-01 No 0.005 mg/ml 5.0 mg/ml 0.15 mg/ml 150 mM buffer Polysorbate 20 Phenol L- L-arginine pH 6.8 Methionine HCl .sup.1pH is adjusted with 0.2N HCl/NaOH
Stability of Target Formulation

(92) Combining all previous results, the good stability was confirmed for the above formulation, as follows:

(93) TABLE-US-00022 TABLE 18 FSH immunoassay Stability results of FSH immunoassay during storage for 3 months at 25 ± 2° C./60 ± 5% relative humidity (RH) and 12 months at 5 ± 3° C.: Results of FSH immunoassay during storage at 25 ± 2° C./60 ± 5% RH, and 12 months at 5 ± 3° C. 25° C. ± 2° C./ 60 ± 5% RH 5° C. ± 3° C. FSH [% of initial] 1 2 3 3 12 Formulation Initial month months months months months J-01 100 95.2 95.9 98.8 96.8 100.5

(94) The results confirm the stability as determined by FSH immunoassay.

(95) TABLE-US-00023 TABLE 19 hCG immunoassay Stability results of hCG immunoassay during storage for 1 month at 25 ± 2° C./60 ± 5% RH is listed in Table 19 and 12 months at 5 ± 3° C. Results of hCG immunoassay during storage at 25 ± 2° C./60 ± 5% RH and 12 months at 5 ± 3° C. 25° C. ± 2° C./ hCG [% of initial] 60 ± 5% RH 5° C. ± 3° C. Formulation Initial 1 month 3 months 12 months J-01 100 88.3 94.1 89.5

(96) The results confirm the stability as determined by hCG immunoassay.

(97) TABLE-US-00024 TABLE 20 Oxidized proteins Stability results of amount of oxidized proteins [% increase from initial] during storage for 3 months at 25 ± 2° C./ 60 ± 5% RH and 12 months at 5 ± 3° C. Results of oxidized proteins during storage at 25 ± 2° C./ 60 ± 5% RH and 12 months at 5 ± 3° C. Oxidized proteins [% increase from initial] 25° C. ± 2° C./60 ± 5% RH 5° C. ± 3° C. Formulation 1 month 2 months 3 months 3 months 12 months J-01 1.8 5.7 7.9 2.6 11
pH

(98) Stability results of pH during storage for 3 months at 25±2° C./60±5% RH and 12 months at 5±3° C. show a stable pH.

(99) TABLE-US-00025 TABLE 21 Results of pH during storage at 25 ± 2° C./60 ± 5% RH and 12 months at 5 ± 3° C. 25° C. ± 2° C./ 60 ± 5% RH 5° C. ± 3° C. pH 1 2 3 3 12 Formulation Initial months months months months months J-01 6.9 6.7 6.8 6.7 6.7 6.8

Example 4

(100) In addition to the above, the present inventors also performed a bioassay study (6 months storage at 25° C. for the DoE batches).

(101) FSH Bioassay (Steelman-Pohley)

(102) Stability results of the FSH bioassay during storage for 6 months at 25±2° C./60±5% RH are listed in Table 22.

(103) TABLE-US-00026 TABLE 22 Results of FSH bioassay during storage at 25 ± 2° C./60 ± 5% RH. The full description of all formulations is given in Table 11. FSH [% of stated 25° C. ± 2° C./ potency (600 IU/ml)] 60 ± 5% RH Formulation 6 months D-01 98.8 D-02 100.7 D-03 102.0 D-04 104.7 D-05 103.4 D-06 101.0 D-07 102.6 D-08 104.9

(104) The results confirm the stability, as determined by FSH bioassay.

(105) LH Bioassay (Seminal Weight Gain)

(106) Stability results of LH bioassay during storage for 6 months at 25±2° C./60±5% RH is listed in Table 23.

(107) TABLE-US-00027 TABLE 23 Results of LH bioassay during storage at 25 ± 2° C./60 ± 5% RH. The full description of all formulations is given in Table 11. LH [% of stated 25° C. ± 2° C./ potency (600 IU/ml)] 60 ± 5% RH Formulation 6 months D-01 87.8 D-02 101.3 D-03 90.9 D-04 95.6 D-05 97.5 D-06 95.0 D-07 97.3 D-08 100.1

(108) The results confirm the stability, as determined by LH bioassay.

(109) Furthermore, the composition of Example 3 was also tested in an FSH and LH bioassay, with the following results:

(110) Stability results of FSH bioassay during storage for 3 months at 25±2° C./60±5% RH and 12 months at 5±3° C. are listed in Table 24:

(111) TABLE-US-00028 TABLE 24 Results of FSH bioassay during storage at 25 ± 2° C./ 60 ± 5% RH and 12 months at 5 ± 3° C. FSH [% of 25° C. ± 2° C./ stated potency 60 ± 5% RH 5° C. ± 3° C. (625 IU/ml)] 1 2 3 3 12 Formulation Initial month months months months months J-01 94.7 93.6 94.5 97.6 95.8 96.1

(112) The results confirm the stability determined by FSH bioassay.

(113) Stability results of LH bioassay during storage for 3 months at 25±2° C./60±5% RH and 12 months at 5±3° C. is listed in Table 25.

(114) TABLE-US-00029 TABLE 25 Results of LH bioassay during storage at 25 ± 2° C./ 60 ± 5% RH and 12 months at 5 ± 3° C. LH [% of stated 25° C. ± 2° C./ potency 60 ± 5% RH 5° C. ± 3° C. (625 IU/ml)] 1 2 3 3 12 Formulation Initial month months months months months J-01 102.1 102.2 99.0 100.0 101.5 100.0

(115) The results confirm the stability determined by LH bioassay.

8 Abbreviations and Definitions

(116) BP British Pharmacopeia DoE Design of experiment DS Drug substance FSH Follicle-stimulating hormone hCG Human chorionic gonadotropin hMG Human menopausal gonadotropin hMG-HP Human menopausal gonadotropin-Highly purified JP Japanese Pharmacopeia LH Luteinizing hormone MD Multiple dose NF National Formulary Ph Eur European Pharmacopeia PS 20 Polysorbate 20 USP U.S. Pharmacopeia WFI Water for injection