IE54374B1

IE54374B1 - Crystalline human leukocyte interferon

Info

Publication number: IE54374B1
Application number: IE2882/82A
Authority: IE
Original assignee: Hoffmann La Roche
Priority date: 1981-12-07
Filing date: 1982-12-06
Publication date: 1989-09-13
Also published as: EP0083734B1; NZ202698A; DK159164C; IL67411A0; AU551407B2; EP0083734A1; CA1209038A; ATE18410T1; JPH0477000B2; AU9115582A; ZA828917B; JPS58164597A; DE3269727D1; DK538482A; IE822882L; DK159164B; PH20413A

Abstract

(For the Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE) 1. Human leukocyte interferon (IFN-alpha in crystalline form. (For the Contracting State AT) 1. A process for the manufacture of human leukocyte interferon in crystalline form, characterized by treating an aqueous solution of the interferon at a concentration of at least about 0.2 mg/ml with polyethylene glycol (PEG).

Description

Human leukocyte interferons designated a^, a.^, 8^, ?>2> P3> Υρ ύ2' ύ3' γ4 and ύ5 ^era-ved from virus induced normal or leukemic donors’ leukocytes have been purified to homogeneity, see U.S. Patent No. 4 289 690, issued September 15, 1981. More recently, recombinant DNA technology has been employed to occasion the microbial production of a number of different human leukocyte interferons (IFN-a) the amino acid sequences of which exhibit in the order of 70 percent or more homology, one relative to another as disclosed in Nature 290, 20-26 (1981). specific recombinant human leukocyte interferons (rIFN-α) described in this publication are rIFN-αΑ, b, C, D, E, F, G and H. Additionally, rIFN-αΙ and rIFN-aJ have been disclosed in German Offenlegungsschrift No. 31 25 706. Furthermore, German Offenle15 gungsschrift No. 31 44 469 describes the microbial production via recombinant DNA technology of hybrid leukocyte interferons. Examples of such hybrid leukocyte interferons include rXFN-α A^_g^ D 93-166Dl-92 A92-165'Al-62 °64 20 °l-63 A63-165;Al-91B93-166’ Al-91F93-166'Al-91 G93-166;Al-150 ^151-165* Bl-92 A92-165;Bl-92 °93-166;Bl-92F93-166' Bl-92G93-166'Dl-92 B93-166;Dl-92F93-166: Dl-92G93-166’Fl-92 A92-165’Fl-92B93-166’ Fl-92D93-166; 25Fl-92 G93-166’ and I 1-151 A152-166‘ The aforesaid natural, recombinant and hybrid human leukocyte interferons represent a family of proteins characterized by a potent ability to confer a virus-resistant state in their target cells. In addition, these interferons can act to inhibit cell proliferation and modulate immune response. These properties have prompted the initial cli54374 nical use of rIFN-αΑ and rlFN-aD as therapeutic agents for the treatment of viral infections and neoplastic diseases.

Crystallization of a substance satisfies one of the classical criteria for homogeneity. Additionally, the crystallization process itself can provide a useful purification step. The availability of large ordered crystals of human leukocyte interferons will also allow the determination of the molecule's tertiary structure utilizing X-ray diffraction.

Numerous techniques have been developed for the crystallization of proteins, however, no generalized procedure has been discovered, and many proteins remain uncrystallized. Thus, crystallization of proteins is an unpredictable art utilizing trial and error procedures among many possible alternative methodologies.

One of the most widely used approach involves the addition to the aqueous protein solution of a crystallizing agent, which is commonly a salt, such as ammonium sulfate or ammonium citrate or an organic solvent, such as ethanol or 2-methyl-2,4-pentanediol. However, such procedures do not provide a suitable means for producing crystalline human leukocyte interferons.

A versatile crystallizing agent is polyethylene glycol (PEG), which combines some of the characteristics of the salts and the organic solvents. See in this regard K.B. Ward et ai., J. Mol. Biol. 98, 161 (1975) and A. McPherson, Jr., J. Biol. Chem. 251, 6300 (1976). It has now been discovered in accordance with the present invention that polyethylene glycol and particularly polyethylene glycol 4000 can be successfully used to crystallize the aforementioned human leukocyte interferons, most particularly rIFN-aA.

Thus, the present invention provides human leuko54374 cyte interferon in crystalline form, pharmaceutical compositions containing the same and a process for the production of said IFN-α which process comprises treating an aqueous solution containing IFN-α at a concentration of at least about 0.2 mg/ml with polyethylene glycol.

The human leukocyte interferons employed as starting materials in the present crystallization process can be isolated by procedures providing the compounds in an essentially homogeneous state. Such procedures include high performance liquid chromatography (HPLC) such as described in the aforesaid U.S. Patent No. 4 289 690, affinity chromatography utilizing a monoclonal antibody to human leukocyte interferon supported on a column support material as described, for example, by T. Staehelin et al., J. Biol.

Chem. 256, 9750-9754 (1981), or any other procedure providing human interferon in sufficient purity (>95%) and in sufficient concentration (^0.2 mg/ml interferon).

There are a number of important advantages attendant to being able to obtain human leukocyte interferons in crystalline form. As indicated above one evident advantage is the additional purification available from the crystallization step which could remove different impurities than the HPLC or conventional column chromatography can achieve. Moreover, crystalline human leukocyte interferons can be stored and shipped at ambient temperatures free of the risk of protease contamination possible in solution storage. Other techniques for producing proteins such as lyophilization are known to cause some denaturation of interferon as evidenced by a loss in the value of the specific acti30 vity of samples before and after such procedures.

A suitable procedure for the crystallization of one of the human leukocyte interferons, rlFN-αΑ is set forth below in the Example. In analogous fashion the other human leukocyte interferons which exhibit a high degree of se35 quence homology can be crystallized. 5437 4 Example rIFN-αΑ (4 ml, 0.2 mg/ml) was dialyzed overnight at 4°C against 0.01 M aqueous HEPES (N-2-hydroxyethylPiperazineΝ' -2-ethane sulfonic acid), adjusted to pH 7.1 with NH^OH.

The solution was concentrated fivefold by evaporation, redialyzed against 0.01 M HEPES (pH 7.1), and further concentrated to 5 mg/ml. The final concentration was determined by ultraviolet spectrophotometry.

A nine-well glass spot plate was siliconized prior to use by dipping it in 5% (v/v) dimethyldichlorosilane in carbon tetrachloride, and then baking it at 180°C. It was washed with water and rebaked. In each of four wells, ul of the interferon solution was placed. A PEG 4000 solution (200 mg/ml) containing NaN^ (0.5 mg/ml) was added to each well to give final concentrations of 20, 40, 60, and 100 mg/ml. The droplets were immediately mixed using a micropipettor, and the spot plate was placed above a solution of 100 mg/ml PEG 4000 contained in a crystallizing dish. The dish was sealed and kept at 4°C. After 1-3 days, crystals appeared in each of the wells. After several more days, larger crystals appeared in some of the droplets.

The crystals were separated from the liquid phases by centrifugation, washed with 0.05 M HEPES (pH 7.1) containing 10% PEG 4000, and redissolved in 0.05 M HEPES, pH 7.1. Bioassays of the solutions prepared from the 10% PEG 4000 mixture revealed that the crystals contained interferon activity. Most of the interferon activity (>90%) was recovered in the crystals. The crystallized protein was indistinguishable from the uncrystallized rIFN-αΑ by gel electrophoresis, which confirms that the crystalline product is uncleaved and intact. 34374 Table RECOVERY OF INTERFERON IN CRYSTALS Crystals Supernatant Crystallization condition Antiviral activity (units/ml) Percentage recovery Antiviral activity (units/ml) Percentage recovery pll 5 4.0 x 108 (94) 2.5 x I07 (6) pH 7 6.0 x 10® (99) 4.5 x 106 (1) pH 8 8.0 x 108 (99) 4.5 x 106 (1) It is not necessary to crystallize interferon A from 10 concentrated solutions. Microcrystals appear in solutions of 0.2 mg/ml interferon, 10% PEG. This method thus may be useful for concentrating dilute solutions of interferon. As is evident from the above Table crystallizing under slightly basic conditions i.e., about pH 8, produces crystalline rIFN-aA in quantitative yield and with the highest specific activity.

Human leukocyte interferon in crystalline form can be utilized in the same manner in pharmaceutical compositions as the previously employed and described purified homo20 geneous interferons, i.e. natural, recombinant and hybrid recombinant human leukocyte interferons respectively.

Claims

1. Human leukocyte interferon (IFN-α) in crystalline form. 2. Interferon in accordance with claim 1, which is natural IFN-a. 3. Interferon in accordance with claim 1, which is recombinant IFN-a. 4. Interferon in accordance with claim 1, which is a hybrid recombinant IFN-a.

2. 5. Recombinant human leukocyte interferon A (rIFN-αΑ) in crystalline form.

3. 6. Interferon in accordance with any one of claims 1-5 as a pharmaceutically active substance.

4. 7. A process for the manufacture of human leukocyte interferon in crystalline form comprising treating an aqueous solution of the interferon at a concentration of at least about 0.2 mg/ml with polyethylene glycol (PEG).

5. 8. A process in accordance with claim 7, wherein the interferon is natural IFN-a.

6. 9. A process in accordance with claim 7, wherein the interferon is a recombinant IFN-a.

7. 10. A process in accordance with claim 7, wherein the interferon is a hybrid recombinant IFN-a.

8. 11. A process in accordance with claim 7, wherein the interferon is recombinant human leukocyte interferon (rIFN-αΑ).

9. 12. A process in accordance with claim 11, wherein PEG 4000 is used and its final concentration is 20-100 mg/ml.

10. 13. A process in accordance with claim 12, wherein the pH value of the solution during the crystallization is about 8.0. 5437 4 δ

11. 14. A process in accordance with claim 12, wherein the concentration of the interferon in the starting solution is about 5.0 mg/ml.

12. 15. A pharmaceutical preparation containing human 5 leukocyte interferon in crystalline form as the active ingredient in association with a pharmaceutically acceptable carrier of diluent therefor.

13. 16. Human leukocyte interferon in crystalline form whenever prepared according to a process claimed in any 10 one of claims 7-14 or by an obvious chemical equivalent thereof.

14. 17. Human leukocyte interferon according to claim 1, substantially as hereinbefore described with particular reference to the accompanying Example. 15

15. 18. Recombinant human leukocyte interferon according to claim 5, substantially as hereinbefore described.

16. 19. A process according to claim 7, for the manufacture of human leukocyte interferon in crystalline

17. 20 form, substantially as hereinbefore described with particular reference to the accompanying Example. 20. Human leukocyte interferon in crystalline form whenever manufactured by a process claimed in a preceding claim.

18. 25 21. A pharmaceutical preparation according to claim 15, substantially as hereinbefore described.