NZ202698A - Human leukocyte interferon in crystalline form - Google Patents

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

New Zealand Paient Spedficaiion for Paient Number £02698 202698 Priority Date(s): l.~. J-?..I Complete Specification Filed: 3.~.Q Class: 0.<51 Publication Date: .... 1? A .1^5.... P.O. Journal. No: .JcfcJC1 NO DRAWINGS NEW ZEALAND PATENTS ACT, 1953 No.

Date: COMPLETE SPECIFICATION CRYSTALLINE HUMAN LEUKOCYTE INTERFERON */We, F. HOFFMANN-LA ROCHE & CO. AKTIENGESELLSCHAFT, 124-184 Grenzacherstrasse, Basle, Switzerland, a Swiss company, hereby declare the invention for which £ / we pray that a patent may be granted to rft¥/us, and the method by which it is to be performed, to be particularly described in and by the following statement: - (followed by la) 202698 - la - Human leukocyte interferons designated a^, a^, 3^, (3_, f},, y, < Tn, Y,, Y. and yc derived from virus induced L i 1 i 3 4 b normal or leukemic donors ' leukocytes have been purified to homogeneity, see New Zealand Patent Specification No. 192201. : More recently, recombinant DNA techno- logy 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 15 human leukocyte interferons (rIFN-a) described in this publication are rIFN-aA, B, C, D, E, F, G and H. Additionally, rIFN-al and rIFN-aJ have been disclosed in New Zealand Patent Specification No. 197572. Furthermore, New Zealand Patent Specification No. 198906 describes the microbial production 20 via recombinant DNA technology of hybrid leukocyte interferons. Examples of such hybrid leukocyte interferons include kl-62 D64-166 ' °l-63 A 6 3 -16 5 ' Al-91 B93-166 ' Al-91 FS rlFN- a A^ 91 D 93-166' °l-92 A92-165 / D1 — 6 3 A63- 165 ' A1 -9 1 B93-166 ' Al-91 F A1 - 9 1 G 9 3 - 166 ' Al-150 I151-165' Bl- 92 B1 — 9 2 °93- 166 ' Bl-92 F9 3-166 ' Bl-92 G D1 -9 2 B93- 166 ' °l-92 F93-166' Dl-92 G F1 -9 2 A92- 165 ' Fl-92 893-166' Fl-92 D Fl-92 G9 3- 166 ' and I -151 A15 2-166" 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 35 can act to inhibit cell proliferation and modulate immune response. These properties have prompted the initial cl-ir-jr^s ° o'- '"S* 3 1 OCT 1935' 2026 nical use of rIFN-aA and r'IFN-aD as therapeutic agents for the treatment of viral infections and neoplastic diseases.

Crystallization of a substance satisfies one of the 5 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 "10 x-ray diffraction.

Numerous techniques have been developed for the crystal lization of proteins, however, no generalized procedure has been discovered, and many proteins remain uncrystallized. 15 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 20 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 25 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 30 K.B. Ward et al. , J. Mol. Biol. 9jB, 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 35 aforementioned human leukocyte interferons, most particularly rIFN-aA.

Thus, the present invention deals with human leuko fr'"* 202698 cyte interferon in crystalline form, with pharmaceutical compositions containing the same and with a method for the production of said IFN-a which method comprises treating an aqueous solution containing IFN-a at a concentration of at least substantially 0.2 mq/iul with polyethylene glycol.

The human leukocyte interferons employed as starting materials in the instant 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 New Zealand Patent Specification No. 192201, 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 (5.0. 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 lyophili-zation are known to cause some denaturation of interferon as evidenced by a loss in the value of the specific activity of samples before and after such procedures.

A suitable procedure for the crystallization of one of the human leukocyte interferons, rIF\'-aA is set forth below in the Example. In analogous fashion the other human leukocyte interferons which exhibit a high degree of se-quence homology can be crystallized. , r »■; 1- WCi I985 2026 Example rIFN-aA (4 ml, 0.2 mg/ml) was dialyzed overnight at 4°C against 0.01 M aqueous HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid), adjusted to pH 7.1 with NH^OH. The solution was concentrated fivefold by evaporation, rcdialyzed 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, 20 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 micropipet tor, 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. Bioassavs 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 uncrystal1ized rIFN-aA by gel electrophoresis, which confirms that the crystalline product is uncleaved and intact. 202698 Table RECOVERY OF INTERFERON IN CRYSTALS Crystals Supernatant Cr ys tal 1 i ?.a t ion condition Antiviral activity (units/ml) Percentage recovery Antiviral activity (units/ml) Percentage recovery pH 5 4.0 x |co ( o (94) 2.5 x 107 (6) pH 7 6.0 x (99) 4.5 x 106 (1) pH 8 00 o X 108 (99) 4.5 x 106 (1) It is not necessary to crystallize interferon A from 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.,. substantially 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 homogeneous interferons, i.e. natural, recombinant and hybrid recombinant human leukocyte interferons respectively. oil "I OCT 1985 yi 202698

Claims (13)

WHAT WE CLAIM IS:

1. Human leukocyte interferon in crystalline form.

2. The crystalline human leukocyte interferon of claim 1 selected from natural, recombinant and hybrid recombinant human leukocyte interferons.

3. The crystalline human leukocyte interferon of claim 1 which is a recombinant human leukocyte interferon.

4. The crystalline human leukocyte interferon of claim 3 which is recombinant human leukocyte interferon A (as hereinbefore defined).

5. A method for the production of human leukocyte interferon in crystalline form which method comprises treating an aqueous solution containing human leukocyte interferon at a concentration of at least substantially 0.2 mg/ml with polyethylene glycol.

6. The method of claim 5 wherein said human leukocyte interferon is selected from natural, recombinant and hybrid recombinant human leukocyte interferons.

7. The method of claim 5 wherein said human leukocyte interferon is recombinant human leukocyte interferon A (as hereinbefore defined).

8. The method of claim 7 wherein said polyethylene glycol (PEG) is PEG 4 000 present in a final concentration of from substantially 20 to 100 mg/ml. . ^ • /■" ••• - ' ^ ' 3 I OCT f985^ 202698 - 7 -

9. The method of claim 8 wherein the pH during crystallization is substantially 8.0.

10. The method of claim 8 wherein the concentration of said interferon for the crystallization procedure is substantially 5.0 mg/ml.

11. A pharmaceutical composition containing as active agent human leukocyte interferon in crystalline form.

12. Human leukocyte interferon in crystalline form whenever prepared according to a method claimed in any one of claims 5 - 10.

13. A method for the production of human leukocyte interferon in crystalline form, substantially as hereinbefore described with particular reference to the foregoing Example. DATED THIS '3 DAY Of 1/^ II ^ ^ A. J. PARK A SON PFR ■ -TC the applicants