WO1995034318A1

WO1995034318A1 - Solution comprising igf-i or any functional analogue thereof and method for its preparation

Info

Publication number: WO1995034318A1
Application number: PCT/SE1995/000685
Authority: WO
Inventors: Jonas Fransson; Ulf Nilsson
Original assignee: Pharmacia & Upjohn Ab
Priority date: 1994-06-16
Filing date: 1995-06-08
Publication date: 1995-12-21
Also published as: AU2757695A; SE9402119D0; IL114063A0

Abstract

The present invention relates to a final drug product comprising IGF-I or any functional analogue thereof in an aqueous solution with a reduced concentration of oxygen. In this way, the IGF-I purity can be retained during storage to a surprisingly high degree. The IGF-I purity can be retained for a prolonged period of time, if the final drug product further comprises an inert gas and/or an antioxidant. The present invention also relates to processes for reducing the oxygen concentration of the aqueous solution, and a method for improving the stability of IGF-I in an aqueous solution by storing the solution under an inert gas atmosphere.

Description

SOLUTION COMPRISING IGF-I OR ANY FUNCTIONAL ANALOGUE THEREOF AND METHOD FOR ITS PREPARATION

Introduction

Insulin-like Growth Factor I (IGF-I) is a peptide present in plasma and other body fluids as well as many cells /tissues. It comprises 70 amino acids, including 3 disulphide bonds, and can stimulate proliferation of a wide range of cell types and it mediates some of the effects of growth hormone. Human IGF-I has been purified from plasma and its complete amino acid sequence is established. (Rinderknecht E et al. "The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin" J. Biol. Chem 253; 2769-76, 1978) Sequences with extensive homologies to human IGF-I are present in IGF-I purified from plasma of other species.

Because of the scarcity of purified plasma IGF-I there was a great necessity to develop methodology for the commercial scale production of IGF-I. Nowadays, such large scale production can readily be achieved by using recombinant DNA techniques. As a result of studies with preparations of recombinant DNA IGF-I it has been demonstrated that it promotes skeletal growth and skeletal muscle protein synthesis. IGF-I has been shown to act both as an endocrine factor as well as a paracrine/autocrine factor. (Skottner et al, Endocrinology, Vol. 124, No 5, 1989 and Cook et al, J Clin Invest 81; 206-212; 1988) Moreover, IGF-I is also effective for the treatment or prevention of catabolic states in patients (Swedish patent application SE 9002731-9) and improves the regeneration of transected peripheral nerves (EP 0308386). It has previously been demonstrated in vitro that IGF-I also can promote actin synthesis in myocytes in culture (Florini, J R, Muscle and Nerve 10 (1987) 577-598 and contractility of neonatal rat cardiocytes in vitro (Vetter, U et al, Basic Res. Cardiol. 83 (1988)647-654).

The stability of proteins is generally a problem in the pharmaceutical industry.

A formulation with a low amount of protein will generally lose activity during purification, sterile manufacturing, storage and during the administration.

It has often been solved by drying of the protein in different drying processes, such as freeze-drying. The protein has thereafter been distributed and stored in dried form. The patient necessarily has to reconstitute the dried protein in a solvent before use, which of course is a disadvantage and is an inconvenience for the patient.

For a patient, who needs daily injections of IGF-I, and especially when the patient is a child, it is of importance that the product is easy to handle, to dose and inject. The reconstitution of a freeze-dried product demands prudence and carefulness and should therefore preferably be avoided.

The freeze-drying process is also a costly and time consuming process step, and it would be of great advantage if this step could be avoided, when preparing a commercial product of a protein.

It would facilitate the use of a pharmaceutical protein if it can be produced and distributed as a stable solution with a prolonged storage life to the patient, who could inject the medicament directly without reconstitution.

Proteins are different with regard to physiological properties. When preparing a pharmaceutical preparation which should be physiologically acceptable, and stable for a long time, consideration can not only be taken to the physiological properties of the protein but also other aspects must be considered such as the industrial manufacture, easy handling for the patient and safety for the patient. The results of these aspects are not predictable when testing different formulations and each protein has often a unique solution regarding stability.

Several solutions have been proposed for the stabilization of different proteins: EP 35204 (Cutter) discloses a method for imparting thermal stability to a protein composition in the presence of a polyol.

EP 381345 (Corint) discloses an aqueous liquid of a peptide, desmopressin, in the presence of carboxymethylcellulose. In WO 89/09614 (Genentech), a stabilized formulation of human growth hormone containing glycine, mannitol, optionally a non-ionic surfactant and a buffer at pH 4-8 is disclosed. The non-ionic surfactant is added for reduced aggregation and denaturation. The formulation has an increased stability in lyophilized form and as a solution obtained after reconstitution. EP 303 746 (International Minerals and Chemical corporation) discloses growth hormone (GH) stabilized in aqueous environment by mixing the growth hormone with polyol, amino acid, polymer of amino acid or choline derivative.

US 4165370 (Coval) discloses a gamma globulin solution and a process for the preparation thereof. The solutions contains polyethylene glycol (PEG).

In EP 77870 (Green Cross) the addition of amino acids, monosaccarides, oligosaccarides or sugar alcohols or hydrocarbon carboxylic acid to improve stability of a solution containing factor NDI is disclosed.

EP 440989 (FUJISAWA) discloses a method for preparing a dried composition of IGF-I, which comprises drying a solution containing IGF-I together with a strong acid.

IGF-I in a citrate buffer at pH 6 is known from WO 91/18621, Genentech. Nothing is mentioned regarding stability of IGF-I.

The patent application PCT /SE94/00010 relates to a stable solution containing Insulin-like Growth factor I (IGF-1) in a phosphate buffer in an amount of 50 mmol or less, giving a pH of 5.5 to 6.5, which is isotonic and suitable for injection.

US 5272135 discloses a method for inhibiting oxidation by adding methionine to a polypeptide. The oxidation process is reduced by the addition of a chemical compound. The concentration of oxygen in the solution is thereby not reduced. US 4727027, Diamond Scientific is directed to a method for photochemical decontamination of aqueous compositions containing biologically active proteins derived from blood or blood components, for minimizing loss in activity. The method comprises adding at least one furocoumarin to the composition and irradiating the obtained composition with ultraviolet (UV) light. Prior to the irradiation, the oxygen concentration of the aqueaous composition can be reduced to inhibit denaturatione.g. by addition of oxygen scavengers, albumins and/or enzyme systems and/or flushing with an inert gas. It is stated in US 4727027 that decreasing the oxygen level has a protecting effect on factor Nm exposed to furocoumarin and UNA light. Noting is mentioned about the stabilising effect on IGF-I in a solution with a reduced concentration of oxygen .

It has now been found that the stability of a solution comprising IGF-I can be further improved if the amount of oxygen is reduced.

Aqueous solutions containing oxygen-sensitive chemical compounds including drugs other than proteins, is normally deoxygenated as follows: Water for injection is bubbled with nitrogen to reduce the concentration of oxygen. The components are dissolved and the solution is bubbled with nitrogen and there-after kept under a nitrogen blanket. During filling, the bottles are flushed with nitrogen gas and the bottles are closed under a stream of nitrogen.

It is, however, not possible to deoxygenate a protein solution by bubbling the solution with a gas. Protein solutions will foam heavily and many protein drugs will denature if exposed to such a treatment. Therefore, it has never been suggested earlier that an aqueous solution containing IGF-I should be stored under an inert gas such as nitrogen.

Figure 1 shows % of oxidized IGF-I during 18 months' storage at 7°C. Figure 2 shows % of oxidized IGF-I during two months' storage at 25°C. Figure 1 shows % of oxidized IGF-I during two months' storage at 50°C. Description of the invention

.We have found that solutions containing IGF-I can be deoxygenated without protein denaturation. Thus, to our great surprise we have found that IGF-I can be in a stable solution, and that such an aqueous solution with a low oxygen content is very stable when stored even at e.g. 25°C.

Thus the present invention relates to a final drug product comprising IGF-I or any functional analogue thereof in an aqueous solution with a reduced concentration of oxygen, for essentially retaining the IGF-I purity and activity during storage.

The oxygen content in the solution can be below 150 mmol/L, suitably below 100 mmol/L and preferably 50 mmol/L or below.

The final product can comprise an inert gas and the solution is suitably stored under the inert gas such as nitrogen, argon or helium, to essentially maintain the low content of oxygen.

The aqueous solution can also contain an antioxidant such as methionine. No concentration or amount can generally be given. It is, however, important that the amount of antioxidant, if used, is in a pharmaceutically acceptable amount For methionine the amount could be e.g. 2 - 50 mmol/L.

The concentration of IGF-I is only dependent of its solubility in the used buffer and the desired therapeutically amount for the given dose. Preferably the concentration of IGF-I is 1-100 mg/ml and more preferably 1-20 mg/ml. The aqueous solution may contain a phosphate buffer, such as sodium phosphate buffer, in an amount of 50 mmol/L or less, e.g.5-20 mmol/L, preferably around 10 mmol/L, giving a pH of 5.5 to 6.5 , preferably 5.7 - 6.2. The solution should be isotonic, which could easily be made by any of several excipients known for a person skilled in the art. E.g. NaCl, glycin, mannitol, glycerol and/or other carbohydrates can be added.

Benzyl alcohol could be chosen as preservative.

The final drug product has less than 2 % increase in oxidized IGF-I after 18 months 'storage at +7±1°C.

It is important that the oxygen concentration is reduced in the solution. In

Example 2 we have shown that it is not enough to reduce oxygen content in the head space in order to retain purity of IGF-I when stored. Also the oxygen concentration in the solution should be reduced.

The invention also refers to a process for preparation of the claimed solution characterised by mixing IGF-I with an aqueous solution, and reducing the oxygen concentration in the solution by subjecting the aqueous solution to an inert gas atmosphere. It can first be done by reducing the pressure and thereafter introducing the inert gas. The latter process is preferably repeated in several cycles.

The invention also refers to a method for improving the stability of IGF-I or any functional analogue thereof in an aqueous solution characterised in that the solution is subjected to a process so that the solution has a reduced concentration of oxygen.

It has been found that the IGF-I solution at a pH of 5.5 to 6.5. shows less than 2 % increase in oxidized IGF-I after storage for at least 18 months at a temperature of 7±l °C

Final drug product relates to the formulated drug in its final container. Suitable containers in the present invention are e.g. vials, syringes and injection devices.

As stated above the low content of oxygen can be essentially maintained by adding an antioxidant to the aqueous solution. The antioxidant must, however, be specifically chosen, as there are few of the known and normally used antioxidants which will function and give the wanted result. Methionine is the preferred antioxidant. It has been found that glutathione, acetylcysteine, sodium bisulfite and ascorbic acid give a decreased stability and EDTA and tocopherol give no effect on stability for IGF-I, see Example 3.

By Insulin-like Growth Factor (IGF-I) is meant both naturally occurring human and animal IGF-I and recombinant IGF-I (rIGF-I), such as rhIGF-I (human), rbIGF-I (bovine) and rpIGF-I (porcine). By functional analogues are meant compounds having the same therapeutic effect as IGF-I in animals and humans.

rhIGF-I is preferred. EXAMPLES

The recombinant human IGF-I (rhIGF-I) used in the experiments was produced in yeast. rhIGF-I was initially synthesised as a hybrid protein fused to the yeast a mating factor pre-pro leader peptide. After expression the primary translation product was secreted out of the cell. During this process the pre-pro-leader was cleaved off. Correctly processed and secreted rhIGF-I could then be isolated from the fermentation media in its native form.

The media with rhIGF-I was then micro filtered and impurities were removed by several chromatographic techniques known within the field.

All buffer components used in the examples fulfil the requirements prescribed in Ph. Eur.2ndEd.

In the example lyophilized IGF-I pools from the final step in the purification process were dissolved in the formulation buffer and chromatographed on a Sephadex G-50 column.

The samples were stored at +7 ± 1°C , +25 ± 1,5°C or +50 ± 1,5°C

Example 1.

This example presents the results from a stability study of a solution which has been stored at +7, +25 and +50°C, respectively.

Nitrogen protection of the solutions was performed by deoxygenation in a vacuum chamber and replacing the evaporated oxygen by nitrogen. The oxygen content was 50 mmol/L.

For comparison, also unprotected (not deoxygenated) solutions were stored. The oxygen content in the unprotected solutions was 300 mmol/L.

COMPOSITION

IGF-I 2 mg Sodium dihydrogen phosphate 5.25 mg Sodium phosphate 0.89 mg

Sodium chloride 6.43 mg

Water for injection to make 1.0 mL This composition gives a concentration of 2 mg/mL IGF-I in 50 mmol/L sodium phosphate buffer, and a pH 6, with 110 mmol/L sodium chloride as tonicity adjuster.

1 mL of this solution was filled in a sterile 8 mL glass vials.

All samples were stored protected from light and investigated after 18 months at

+7 ± 1°C and after 2 months at +25 ± 1,5°C and at +50± 1,5°C.

RESULTS. The results after storage at +5°C, +25°C and +50°C are presented in Figures 1-3.

CONCLUSION

At 7, 25 and 50 °C, respectively it can clearly be seen that the rate of oxidation of IGF-I is reduced when IGF-I is stored in a solution with a reduced concentration of oxygen. At 7°C the purity of the IGF-I solution was even not changed after 18 months storage.

Example 2

This example presents the results from a stability study of solutions filled in glass vials with or without head space and then stored at +5°C± 3°C and +25°C ± 3°C. The head space contained 1.5 mL of sterile air.

The purpose of this experiment was to investigate whether removal of the head space in the vials was sufficient to enhance stability of IGF-I.

COMPOSΓΠON

Composition as in example 1.

RESULTS

Results after storage at +5°C and +25°C are presented in tables 1 and 2.

Table 1 Purity and percentage of oxidized IGF-I. 2 mg/mL IGF-I with or without head space stored at +5°C ± 3°C.

TTME With head space Without head space

Months Purity % ox. IGF-I % Purity % ox. IGF-I %

0 97.4 1,7 97.8 1.6

9 97.1 1.9 97.4 1.9

24 95.0 2.4 95.3 2.5 Table 2 Purity and percentage of oxidized IGF-I.

2 mg/mL IGF-I with or without head space stored at +25°C ± 3°C.

TTME With head space Without head space

Months Purity % ox. IGF-I % Purity % ox. IGF-I %

0 97.4 1.7 97.8 1.6 2 96.1 2.2 96.2 2.1 6 83.5 3.9 83.9 3.6

CONCLUSIONS

Reducing the head space in the vials is not sufficient to improve the stability of IGF-I or to reduce oxidation.

Example 3

This example presents the results from a stability study of solutions to which have been added different antioxidants.

COMPOSITION

IGF-I l mg Sodium dihydrogen phosphate 5.25 mg Sodium phosphate 0.89 mg Sodium chloride 6.43 mg Water for injection to make 1.0 mL

This composition gives a concentration of 1 mg/mL IGF-I in 50 mmol/L sodium phosphate buffer, and a pH 6, with 110 mmol/L sodium chloride as tonicity adjuster. To these solutions were added the following common antioxidants: Ascorbic acid, sodium bisulphite, reduced glutathione, acetylcyteine, methionine, tocopherol, EDTA, 1,4-dithiothreitol, sodium tiosulphate, n- propylgallat and L-tryptophan.

All were added in an amount of 10 mmol/L.

The solutions were filled in glass vials and then stored at +25°± 1°C.

RESULTS

The oxidants sodium bisulphite, reduced glutathione, acetylcysteine, 1,4- dithiothreitol, sodium tiosulphate, n-propylgallat and L-tryptophan were all incompatible with IGF-I. as precipitates of IGF-I were formed after some hours of storage. Ascorbic acid and sodium tiosulphate did in fact increase oxidation rates. EDTA and tocopherol did not enhance the stability towards oxidation. The antioxidant methionine did reduce the oxidation rate during storage. The results after storage are shown in table 3.

Table 3 Purity and percentage of oxidized IGF-I 1 mg/mL IGF-I with or without the added antioxidant methionine after storage at +25°C

TIME With methionine Without methionine

Months Purity % ox. IGF-I% Purity % ox. IGF-I%

0 97.9 1.4 98.0 1.5

2 97.6 1.5 96.9 1.9

CONCLUSIONS

With the exception of methionine, the commonly used antioxidants for pharmaceutical products are not possible to use with the protein drug IGF-I.

Claims

1. Final drug product comprising IGF-I or any functional analogue thereof in an aqueous solution with a reduced concentration of oxygen, for essentially retaining the IGF-I purity and activity during storage.

2. Final drug product according to claim 1, which further comprises an inert gas.

3. Final drug product according to claim 2, in which the inert gas is nitrogen.

4. Final drug product according to any of claims 1-3, in which the aqueous solution further contains an antioxidant .

5. Final drug product according to claim 4, in which the antioxidant is methionine.

6. Final drug product according to any of claims 1-5, in which the concentration of IGF-I is 1-100 mg/ml and more preferably 1-20 mg/ml.

7. Final drug product according to any of claims 1-6, in which the aqueous solution further contains a phosphate buffer in an amount of 50 mmol/L or less, giving a pH of 5.5 to 6.5 , preferably 5.7 - 6.2 in an isotonic solution for injection.

8. Final drug product according to any of claims 1-7, in which the amount of phosphate buffer is 5-20 mmol/L, preferably around 10 mmol/L.

9. Final drug product according to any of claims 1-8, in which the buffer is sodium phosphate buffer.

10. Final drug product according to any of claims 7-9 which further containing an isotonic agent such as NaCl. and optionally a preservative.

11. Final drug product according to claim 10 which contains benzyl alcohol as preservative.

12. Final drug product according to claim 10 which further contains glycine, glycerol, mannitol and/or carbohydrates as isotonic agents.

13. Final drug product according to any of claims 1-13 which after +7±1°C has less than 2 % increase in oxidized IGF-I after 18 months 'storage.

14. Process for preparation of the solution according to any of claims 1-13, characterised by mixing IGF-I with an aqueous solution, and reducing the oxygen concentration in the solution by subjecting the solution to an inert gas atmosphere.

15. Process for preparation of the solution according to any of claims 1-13, characterised by mixing IGF-I with an aqueous solution, and reducing the oxygen concentration by first reducing the pressure and thereafter introducing the inert gas, preferably repeated in several cycles.

16. Method for improving the stability of IGF-I or any functional analogue thereof in an aqueous solution characterised in that the solution is subjected to a process so that the solution has a reduced concentration of oxygen.

17. Method according to claim 16, characterised in that the IGF-I solution at a pH of 5.5 to 6.5 shows less than 2 % increase in oxidized IGF-I after storage for at least 18 months at a temperature of 7±1 °C .