IE60061B1 - Stabilization of interferons - Google Patents

Description

The present invention is concerned with interferon compositions with improved stability of the biological and immunological interferon activity as well as a process for their manufacture.

Under interferons there is to be understood a group of body-specific proteins with antiviral and immunoregulatory activity. The antiviral effect is achieved not by a direct influence on the viruses themselves, but by an activity on their target cells in the sense of a protection against the virus infection. In addition to the antiviral activity, the interferons can exert objectifiable effects on cancer tumours, which makes them suitable for use in cancer therapy, and they influence the immune system of the body in that e.g. they activate macrophages and NK cells and increase the expression of various immunologically significant constituents of the cell membrane.

Thanks to recombinant DNA technology, interferons (IFN-α, -β and -γ) can today be prepared in a microbiological manner in amounts which hitherto could not be made available by isolation from natural material (leucocytes, fibroblasts, lymphocytes) and purification in spite of the greatest efforts.

For the first time this new technology has opened a way for the intensive clinical testing and possible wide therapeutic use of interferons and has made possible an adequate supply of the active substances for persons seeking a treament with the active substances, Among the human interferons IFN-γ is the most unstable protein, the biological activity of which is lost upon storage, freezing or lyophilization. This instability impedes to a considerable extent the manufacture, purification, storage and therapeutic use of IFN-γ. The stabilization of its biological activity therefore plays a «? particularly important role. In comparison to IFN-γ, the human interferons e and β are relatively stable proteins * and can be purified, stored and used in therapy without any loss of biological activity which is worth mentioning.

Various adjuvants have hitherto been used for the stabilization of interferons, especially IFN-γ. Thus, the stabilization of interferons by means of propylene glycol has been described in U.S. Patent Wo. 4 483 849. It is has,, however, emerged that propylene glycol can not be used efficiently for the stabilization of recombinant IFN-γ. The stabilization of IFN-γ by means of bovine serum albumin (Rinderknecht et al.» J. Biol. Chem. 259,, 679O-S797 [1984.]) has also been examined and has been found not to be efficient. The stabilization of IFN-γ by means of 0.1% BSA and 0.5% gelatine (Devos et al.

J. Interferon Res. 4» 451-468 [1984]) is also known. In order to avoid a loss of activity, such preparations must, however, always be stored at -7O°C.

Compositions containing mouse interferon, which contain a phosphate buffer system having a pH value of 7 and a phosphate concentration of O.lM and 0.5% bovine serum albumin, are known from Cryobiology 16, 301-314 (1979).

In accordance with the invention it has now been found that interferons have an excellent stability in the presence of a high concentration of phosphate and/or polyphosphate, as a result of which the interferons can be stored at 2-8°C in solutions without any loss of biological and immunological activity which is worth mentioning.

The present invention is therefore concerned with preparations containing interferon,, a phosphate buffer system and/or polyphosphate,, as well as a process for their manufacture, which comprises treating an interferon 5 solution with phosphate and/or polyphosphate. 1 0 Moreover, the present invention is concerned with pharmaceutical preparations based on the compositions in accordance with the invention, as well as a process for the manufacture of such pharmaceutical preparations, which is characterized by mixing a composition in accordance with the invention and, if desired, one or more other therapeutically valuable substances with a pharmaceutically acceptable carrier and bringing the resulting mixture into a suitable dosage form. 15 The stabilization in accordance with the invention can 20 be used on all interferons or polypeptides with interferon-like biological properties as well as on mixtures which contain an interferon or such a polypeptide. It is preferably used in che case of human interferon. The human interferon can be a natural or 25 recombinant leucocyte interferon (IFN-α), fibroblast interferon (IFN-β) or immune interferon (IFN-γ). So-called hybrid interferons, in which fragments of two or more native interferon species are linked, can be stabilized in accordance with the invention. An especially preferred human interferon in connection with the present 30 fl invention is IFN-γ, preferably recombinant IFN-γ especially IFN-γ DO or IFN-γ D3. The designations DO and D3 are used to differentiate the last-named interferons,, whereby by DO there is denoted that rIFN-γ whose N-terminal amino acid sequence begins with Cys-Tyr-Cys or Met-Cys-Tvr-Cys, while under 03 there is to be understood a rIFN-γ which Is shortened by Cys-Tyr-Cys 35 and whose N-terrainal amino acid sequence begins with Gin or Met-Gln.

The content of interferon in the compositions in accordance with the invention is not critical. The concentration range covers several powers of radix 10 and above this is limited essentially only by the solubility of the interferon in question. For human XFN-γ there come into consideration, for example, concentrations up to 8 units (U)/ral„ with a preferred range being 4·103 - about 10δ U/ml.

The compositions in accordance with the invention can 10 be used for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of viral infections and immunoregulatory anomalies, in particular neoplasms .

The phosphate buffer system of the present invention 15 is arranged so that it maintains a pH of about 6.0-9.0, preferably of 7.5, and has a phosphate concentration of 500-1000 mmol/1, preferably 1000 mmol/1. Potassium phosphates and sodium phosphates, preferably disodium hydrogen phosphate and monosodium hydrogen phosphate, come into consideration as the phosphate source. An especially preferred phosphate buffer system of the present invention is arranged so that it guarantees a pH of about 7.5 and contains a phosphate concentration of 500-1000 mmol/1, preferably 1000 mmol/1, as well as 25-100 g/1, preferably 50 g/1, of polyphosphate, especially potassium polyphosphate.

The polyphosphate solutions of pH 7.5 of the present invention are used in a concentration of 25-100 g/1, preferably 50 g/1, of polyphosphate,, especially potassium polyphosphate.

Particulars of the invention are described in the following Examples.

The chemicals used in the Examples were p.a. or of highest purity.

All interferons stabilized in accordance with the invention, especially the recombinant T.FN-γ D3 used in the Examples, can be obtained in pure form according to known methods described in the literature or according to methods which are familiar to the person skilled in the art.

For the determination of the immunological activity of recombinant IFN-γ D3 there was used an enzyme-immunological test (ϊΡΝ-γ-ΕΙΑ) corresponding to the enzyme- immunological test of H. Gallati for the determination of human leucocyte interferon which is described in J. Clin. Chem. Clin. Biochem. 20, 907-914 (1982).

For the stability testing of the IFN-γ,, solutions with 4000-10000 U/ml of IFN-γ and the additives under investigation were incubated in glass ampoules at 437°c and after fixed time intervals the immunological activity of the IFN-γ still present in the incubated IFN-γ solution was determined with the IFN-γ-ΕΙΑ.

Fig. 1 shows the stability of the immunological activity of the IFN-γ as a function of the buffer system and pH value. 6000 U/ml of IFN-γ were taken up in each case in 0.1 mol/1 of sodium acetate of pH 2.0-6.0 (-), sodium phosphate of pH 5.5-8.0 (»-#) and in TRIS/acetate of pH 7.2-9.0 (A-A) and incubated for 2 days in sealed glass ampoules at 37°C. The immunological activity of the IFN-γ was determined with the IFN-γ-ΕΙΑ prior to the incubation as well as one and 2 days after incubation.

Fig. 2 shows che stability of the immunological activity of the IFN-γ in 500 mmol/1 of sodium phosphate as a function of the pH value. 10000 U/ml of IFN-γ were taken up in 500 mmol/1 of sodium phosphate buffet of pH 4.5-10 and incubated in glass ampoules for 7 days at ·> 37°C. After 3 days (/-·) and after 7 days incubation (ο“ --o) the immunological activity was determined with the IFN-γ-ΕΙΑ.

Fig. 3 shows the stability of che immunological activity of the IFN-γ at the pH value 7.5 as a function of the sodium phosphate concentration. 6000 U/ml of IFN-γ were taken up in solutions which were buffered with different sodium phosphate concentrations and which were adjusted to the pH value 7.5. The IFN-γ solutions were incubated in glass ampoules for 7 days at 37°C. After this incubation the immunological activity in the IFN-γ solutions was determined with the IFN-γ-ΕΧΆ.

Fig. 4 shows the stability of the immunological activity of the IFN-γ as a function of the polyphosphate concentration in the solution. 4500 U/ml of IFN-γ were taken up in solutions which contained various concentrations of potassium polyphosphate, pH 7.5. These IFN-γ solutions were incubated in glass ampoules for IS days at 37°c. After 3 days (*-“·) and after 16 days incubation (o™ 0) the immunological activity of theIFN-γ in the respective solutions was determined with the IFN-γ-ΕΙΆ.

Fig. 5 shows the stability of the immunological activity of the IFN-γ as a function of the polyphosphate concentration in the presence of and in the absence of sodium phosphate. 0000 U/ml of IFN-γ were taken up in solutions which contained different concentrations of potassium polyphosphate of pH 7.5 (o “o). I n the solutions were buffered with addition, a number of 1000 mmol/1 of sodium phosphate of pH 7.5 (* *).

The IFN-γ activities set forth in this Figure were measured with the XFN-γ-ΕΙΑ after a 20 days incubation Fig. 6 shows comparative stability tests with IFN-γ in various solvents and at various temperatures. In each case 4000 U/ml of IFN-γ were taken up in a) 20 mraol/1 of sodium phosphate with 9 g/1 of NaCl and 0.25 g/1 of Thimerosal (sodium ethylmercurithiosalicylate, mfr. Fluka), pH 7.5 (□ □), b) 20 mmol/1 of sodium phosphate with 9 g/1 of NaCl, g/1 of BSA and 0.25 g/1 of Thimerosal, pH 7.5 ( Mi), and c) 1000 mmol/1 of sodium phosphate, 50 g/1 of potassium polyphosphate and 0.25 g/1 of Thimerosal,, pH 7.5 (Δ Δ, ο o,. * ·).

These IFN-γ solutions were incubated in glass ampoules for 8 days at 2-8°C, 22°C and -s-37°C. After fixed time intervals the immunological activity of the IFN-γ was determined with the IFN-γ-ΕΙΆ.

Example 1 In a first series of experiments the IFN-γ stability in various buffer systems and at various pH values was investigated. 8000 U/ml of IFN-γ were taken up in each case in 0.1 mol/1 of sodium acetate of pH 2.0-8.0,, sodium phosphate of pH 5.5-8.0 and in TSXS/acetate of pH 7.2-9.0 and incubated for 2 days in sealed glass ampoules at 37°C The immunological activity of the IFN-γ was determined with the XFN-γ-ΞΙΑ prior to the incubation as well as one and 2 days after incubation. The results presented in Fig. 1 show in general a poor stability of the immuno9 logical activity of the IFN-γ in 0.1 mol/1 of the buffer systems investigated. The best results are achieved with the phosphate buffer system.

Example 2 In a further series the IFN-γ stability in 500 mmol/1 of sodium phosphate as a function of the pH value (4.5-10) was investigated. In each case 10.000 U/ml of IFN-γ were taken up in 500 mmol/1 of sodium phosphate buffer of pH 4.5-10 and incubated in glass ampoules for 7 days at 37°C. After 3 days and after 7 days incubation the immunological activity was determined with the IFN-γ-ΕΙΑ. It was established on the one hand that generally a substantially improved stability can be achieved with the increased phosphate concentration and on the other hand that within the investigated pH range of 4.5-10 the IFN-γ is most stable at pH 7.5 (Fig. 2).

Thus, after a 7 days incubation at 37°C in a solution of pH 7.5 buffered with 500 mmol/1 of sodium phosphate 70% of the original immunological activity of the IFN-γ used could still be found.

Example 3 On the basis of the surprising results from Example 2 the influence of the sodium phosphate concentration at pH 7.5 on the IFN-γ stability at an incubation temperature of --37°C was investigated. 6000 U/ml of IFN-γ were taken up in solutions which were buffered with different sodium phosphate concentrations and adjusted to the pH value 7.5. The IFN-γ solutions were incubated in glass ampoules for 7 days at 37°C. After this incubation period the immunological activity in the IFN-γ solutions was determined with the IF'M-γ-ΕϊΑ. The results, which are compiled in Fig. 3, confirm the ζ » JL findings presented in Fig. 1 and 2 and show a substantial improvement of the IFN-γ stability with increasing phosphate concentration.

Example 4 As the sodium phosphate concentration can not be increased substantially above 1000 mmol/1 on solubility grounds (crystallization-out at 4°C), in a further series of experiments the influence of polyphosphate on the IFN-γ stability at 37°C was investigated. 4500 U/ml of IFN-γ were taken up in solutions which contained various concentrations of potassium polyphosphate, pH 7.5. These IFN-γ solutions were incubated in glass ampoules for 16 days at 37°C. After 3 and after IS days the immunological activity of the IFN-γ in the respective solutions was determined with the IFN-γ-ΕΙΑ. The results (Fig. 4) show a similar good effect as with high concentrations of sodium phosphate.

Example 5 In order to clarify the influence of a combination of potassium polyphosphate and of sodium polyphosphate on the stability of the IFN-γ, 8000 U/ml of IFN-γ were taken up in solutions which contained different concentrations of potassium polyphosphate of pH 7.5. In addition, a number of the solutions were buffered with 1000 mmol/1 of sodium phosphate of pH 7.5. The results (Fig. 5) show an optimal stabilization of IFN-γ with a combination of 1000 mmol/1 of sodium phosphate of pH 7.5 and 25-100 g/1,. preferably 50 g/1, of polyphosphate.

Example a Finally,, the stability of the IFN-γ in diverse solvents was investigated. Thus, in each case 4000 U/ml of il IFN-γ were taken up in (a) 20 mmol/1 of sodium phosphate with 9 g/1 of NaCl and 0.25 g/1 of Thimerosal, pH 7.5, (b) 20 mmol/1 of sodium phosphate with 9 g/1 of NaCl, g/1 of BSA and 0.25 g/1 of Thiraerosal, pH 7.5, and (c) 1000 mmol/1 of sodium phosphate, 50 g/1 of potassium polyphosphate and 0.25 g/1 of Thimerosal, pH 7.5. These IFN-γ solutions were incubated in glass ampoules for 8 days at 2-8°C 22°C and *37°C. After fixed time intervals the immunological activity of the IFN-γ was determined. As is evident from Fig. 6, the comparison of the storage stabilities of the IFN-γ in PBS, in PBS-BSA as well as in 1000 mmol/1 of sodium phosphate/50 g/1 of sodium polyphosphate of pH 7.5 shows that the immunological activity of the IFN-γ in PBS and in PBS-3SA at an incubation temperature of 22°C is almost completely lost even after 1 day, while it remains almost completely in the phosphate-polyphosphate solution after 8 days incubation at 4*C. at 22°C and at 37°C

Claims (5)

Claims

1. A composition containing interferon, a phosphate buffer system, which guarantees a pH value of S,.0-9.0, preferably of 7.5, and which has a phosphate . concentration of 500-1000 mmol/1„ preferably 1000 mmol/1, and/or polyphosphate .

2. A composition according to claim 1, characterized in that the phosphate buffer system contains disodium hydrogen phosphate and monosodium hydrogen phosphate.

3. A composition according to claim 1 or 2, characterized in that it contains 25-100 g/l of polyphosphate .

4. A composition according to claim 1 or 2, characterized in that It contains 50 g/l of polyphosphate. 5. A composition according to claim 1, characterized in that it contains 25-100 g/l of polyphosphate solution of pH 7.5. 5. A composition according to claim 1, characterized in that it contains 50 g/l of polyphosphate solution of pH 7.5. 7, A composition according to claim 5 or 6, characterized in that it contains potassium polyphosphate. 8. A composition according to any one of claims 1-7, characterized in that the interferon is a human interferon. 9. A composition according to claim 8,, characterized in that the human interferon is a natural or a recombinant IFN-γ. 10. A composition according to claim 9, characterized in that It contains rIFN-γ DO. 11. A composition, according to claim 9, characterized in that it contains rIFN-γ D3. 12. A composition containing recombinant human IFN-γ 03,, 1000 mmol/1 of sodium phosphate buffer of pH 7.5 and 50 g/1 of potassium polyphosphate. 13. A composition according to any one of claims 1-12 as a pharmaceutically active agent. 14. A composition according to any one of claims 1-12 as an antiviral and immunoregulatory agent. 15. A process for the manufacture of a composition according to any one of claims 1-12,,, characterized by treating an interferon solution with a phosphate buffer system in accordance with claim 1 and/or polyphosphate. 16. A pharmaceutical preparation based on a composition according co any one of claims 1-12. 17. The use of a phosphate buffer system according to claim 1 and/or of polyphosphate for the stabilization of interferon. 18. The use of a composition according to any one of claims 1-12 for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of diseases. 19. The use of a composition according to any one of claims 1-12 for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of viral infections . 20. The use of a composition according to any one of claims 1-12 for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of immunoregulatory anomalies. 21. The use of a composition according to any one of claims 1-12 for the manufacture of a pharmaceutical preparation for the therapy or prophylaxis of neoplasms. 22. A composition according to Claim 1, substantially

5. As hereinbefore described and exemplified. F. R. KELLY δ CO.,