CA1209038A - Crystalline human leukocyte interferon - Google Patents
Crystalline human leukocyte interferonInfo
- Publication number
- CA1209038A CA1209038A CA000417034A CA417034A CA1209038A CA 1209038 A CA1209038 A CA 1209038A CA 000417034 A CA000417034 A CA 000417034A CA 417034 A CA417034 A CA 417034A CA 1209038 A CA1209038 A CA 1209038A
- Authority
- CA
- Canada
- Prior art keywords
- human leukocyte
- interferon
- leukocyte interferon
- polyethylene glycol
- crystalline form
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/56—IFN-alpha
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Abstract Human leukocyte interferon in crystalline form is described as well as a method for its production by treating an aqueous solution containing it with poly-ethylene glycol.
Description
~Z~3~
~luman leukocyte interferons designated a1, a2~
~2' ~3~ Yl, Y2, y3, y4 and y5 derived from virus induced normal or leukemic donors' leukocytes have been purified to homogeneity, see U.S. Pa-tent No. ~ 289 690, issued September 15, 1981. More recently, recombinant DNA techno-10 logy has been employed to occasion the microbial production of a number of different human leukocyte interferons (IFN-a) the amino acid se~uences of which exhibit in the order of 70 percent or more homology, one relative to another as disclosed in Nature 290, 20-26 (1981)o Specific recombinant 15 human leukocyte interferons (rIFN-a) described in this publication are rIFN-aA, ~, C, D, E, F, G and H. Additionally, rIFN-aI and rIFN-aJ have been disclosed in German Offenle-gungsschrift No. 31 25 706. Furthermore, German Offenle-gungsschrift No. 31 44 469 describes the microbial pro-20 duction via recombinant DNA technology of hybrid leukocyteinterferons. Examples of such hybrid leukocyte interferons include rIFN-a Al-91 D93-166i Dl-92 A92-165i Al-62 D64-166;
Dl-63 A63-165i Al--91 B93--166i Al-91 93-166i 1-91 93-166; 1-150 I151-165; Bl-92 A92-165 Bl-92 D93-166; Bl-92 F93-166; Bl-92 G93-166i Dl-92 B93-166i Dl-92 F93-166i 1-92 93-166;
Fl-92 A92-165i Fl-92 B93-166i Fl-92 D93-166 Fl 92 G93 166i and Il_l51 A152_166 The aforesaid natural, recombinant and hybrid human leukocyte inter~erons represent a family of proteins charac-terized 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 cli-Mez/26.10.82 nical use of rIFN-aA and rIFN-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 crystalliza-tion process itself can provide a useful puri-fication step. The availability of large ordered crystals of human leukocyte interferons will also allow the deter-mination of the molecule's tertiary structure u-tilizing 10 X-ray di~fraction.
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 crystalli-zing 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 pro-cedures do not p~ovide 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 3~ K.B. Ward et al., J. Mol. Biol. 98, 161 (1975) and A. ~cPherson, 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 parti-cularly rlFN-aA.
Thus, the present invention deals with human leuko-~Z~ 3~3 cyte interferon in crystalline form, wi-th 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 about 0.2 mg/ml with polyethylene glycol.
The human leukocyte interferons employed as starting materials in the instant crystallization process can be iso-lated by procedures providing the compounds in an essen-10 tially homogeneous state. Such procedures include highperformance liquid chromatography (HPLC) such as described in the aforesaid U.S. Patent No. 4 289 690, affinity chro-matography utilizing a monoclonal antibody to human leuko-cyte interferon supported on a column support material as 15 described, for example, by T. Staehelin et al., J. Biol.
ChemO 256, 9750-9754 (1981), or any other procedure provi-ding 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 crystal-lization step which could remove different irnpurities than 25 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-30 zation are known to cause some denaturation of interferonas evidenced by a loss in the value of the specific acti-vity of samples before and after such procedures.
A suitable procedure for the crystallization of one of 35 the human leukocyte interferons, rIFN-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.
3~
Thus the present inventi~n provides a method for the production o~ recombinant human leukocyte interferon A in crystalline ~oxm which method comprises treating an aqueous solution containing recombinant human leukocyte interferon A
at a concentration of at least 0.2 mg/ml with polyethylene glycol to a final concentration o~ polyethylene glycol of from about 20 to lO0 mg/ml.
Ex~mple rIFN-aA (4 ml, 0.2 mg/ml) was dialyzed overnight at 4C against 0.01 M aqueous HEPE~ (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid), adjusted to pH 7.1 with NH40H.
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 180C. It was washed with water and rebaked. In each of four wells, 20 ~1 of the interferon solution was placed. A PEG 4000 solution (200 mg/ml) containing NaN3 (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 solu-tion of 100 mg/ml PEG 4000 contained in a crystallizingdish. The dish was sealed and kept at 4C. After 1-3 days, crystals appeared in each of the wells. ~fter 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 re-covered in the crystals~ The crystallized protein wasindistin~uishable from the uncrystallized rIFN-aA by ~el electrophoresis, which confirms that the crystalline product is uncleaved and intact.
~2~3i3~
_ 5 Table .
RECOVERY OF INTERFERON IN CRYSTALS
Crystals Supernatant Crystallization Antiviral activity Percentage Antiviral Percentage condition ~units/ml) recovery activity recovery (units/ml) ~0 pH 5 4.0 x 10 (94) 2.5 x 10 (6) pH 7 6.0 x 10 (99) 4.5 x 10 (1) pH 8 8.0 x 10 (99) 4.5 x 10 (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.
20 As is evident from the above Table crystalllzing 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 homo-geneous interferons, i.e. natural, recombinant and hybrid re~ombinant human leukocyte interferons respectively.
~luman leukocyte interferons designated a1, a2~
~2' ~3~ Yl, Y2, y3, y4 and y5 derived from virus induced normal or leukemic donors' leukocytes have been purified to homogeneity, see U.S. Pa-tent No. ~ 289 690, issued September 15, 1981. More recently, recombinant DNA techno-10 logy has been employed to occasion the microbial production of a number of different human leukocyte interferons (IFN-a) the amino acid se~uences of which exhibit in the order of 70 percent or more homology, one relative to another as disclosed in Nature 290, 20-26 (1981)o Specific recombinant 15 human leukocyte interferons (rIFN-a) described in this publication are rIFN-aA, ~, C, D, E, F, G and H. Additionally, rIFN-aI and rIFN-aJ have been disclosed in German Offenle-gungsschrift No. 31 25 706. Furthermore, German Offenle-gungsschrift No. 31 44 469 describes the microbial pro-20 duction via recombinant DNA technology of hybrid leukocyteinterferons. Examples of such hybrid leukocyte interferons include rIFN-a Al-91 D93-166i Dl-92 A92-165i Al-62 D64-166;
Dl-63 A63-165i Al--91 B93--166i Al-91 93-166i 1-91 93-166; 1-150 I151-165; Bl-92 A92-165 Bl-92 D93-166; Bl-92 F93-166; Bl-92 G93-166i Dl-92 B93-166i Dl-92 F93-166i 1-92 93-166;
Fl-92 A92-165i Fl-92 B93-166i Fl-92 D93-166 Fl 92 G93 166i and Il_l51 A152_166 The aforesaid natural, recombinant and hybrid human leukocyte inter~erons represent a family of proteins charac-terized 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 cli-Mez/26.10.82 nical use of rIFN-aA and rIFN-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 crystalliza-tion process itself can provide a useful puri-fication step. The availability of large ordered crystals of human leukocyte interferons will also allow the deter-mination of the molecule's tertiary structure u-tilizing 10 X-ray di~fraction.
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 crystalli-zing 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 pro-cedures do not p~ovide 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 3~ K.B. Ward et al., J. Mol. Biol. 98, 161 (1975) and A. ~cPherson, 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 parti-cularly rlFN-aA.
Thus, the present invention deals with human leuko-~Z~ 3~3 cyte interferon in crystalline form, wi-th 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 about 0.2 mg/ml with polyethylene glycol.
The human leukocyte interferons employed as starting materials in the instant crystallization process can be iso-lated by procedures providing the compounds in an essen-10 tially homogeneous state. Such procedures include highperformance liquid chromatography (HPLC) such as described in the aforesaid U.S. Patent No. 4 289 690, affinity chro-matography utilizing a monoclonal antibody to human leuko-cyte interferon supported on a column support material as 15 described, for example, by T. Staehelin et al., J. Biol.
ChemO 256, 9750-9754 (1981), or any other procedure provi-ding 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 crystal-lization step which could remove different irnpurities than 25 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-30 zation are known to cause some denaturation of interferonas evidenced by a loss in the value of the specific acti-vity of samples before and after such procedures.
A suitable procedure for the crystallization of one of 35 the human leukocyte interferons, rIFN-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.
3~
Thus the present inventi~n provides a method for the production o~ recombinant human leukocyte interferon A in crystalline ~oxm which method comprises treating an aqueous solution containing recombinant human leukocyte interferon A
at a concentration of at least 0.2 mg/ml with polyethylene glycol to a final concentration o~ polyethylene glycol of from about 20 to lO0 mg/ml.
Ex~mple rIFN-aA (4 ml, 0.2 mg/ml) was dialyzed overnight at 4C against 0.01 M aqueous HEPE~ (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid), adjusted to pH 7.1 with NH40H.
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 180C. It was washed with water and rebaked. In each of four wells, 20 ~1 of the interferon solution was placed. A PEG 4000 solution (200 mg/ml) containing NaN3 (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 solu-tion of 100 mg/ml PEG 4000 contained in a crystallizingdish. The dish was sealed and kept at 4C. After 1-3 days, crystals appeared in each of the wells. ~fter 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 re-covered in the crystals~ The crystallized protein wasindistin~uishable from the uncrystallized rIFN-aA by ~el electrophoresis, which confirms that the crystalline product is uncleaved and intact.
~2~3i3~
_ 5 Table .
RECOVERY OF INTERFERON IN CRYSTALS
Crystals Supernatant Crystallization Antiviral activity Percentage Antiviral Percentage condition ~units/ml) recovery activity recovery (units/ml) ~0 pH 5 4.0 x 10 (94) 2.5 x 10 (6) pH 7 6.0 x 10 (99) 4.5 x 10 (1) pH 8 8.0 x 10 (99) 4.5 x 10 (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.
20 As is evident from the above Table crystalllzing 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 homo-geneous interferons, i.e. natural, recombinant and hybrid re~ombinant human leukocyte interferons respectively.
Claims (6)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for the production of recombinant human leukocyte interferon A in crystalline form which method comprises treating an aqueous solution containing recombinant human leukocyte interferon A at a concentration of at least 0.2 mg/ml with polyethylene glycol to a final concentration of polyethylene glycol of from about 20 to 100 mg/ml.
2. The method of claim 1 wherein the polyethylene glycol (PEG) is PEG 4000.
3. The method of claim 2 wherein the pH during crystallization is about 8Ø
4. The method of claim 2 wherein the concentration of said interferon to be crystallized is about 5.0 mg/ml.
5. Recombinant human leukocyte interferon A in crystalline form whenever prepared according to the process claimed in claim 1 or by an obvious chemical equivalent thereof.
6. Recombinant human leukocyte interferon A in crystalline form whenever prepared according to a process claimed in any one of claims 2-4 or by an obvious chemical equivalent thereof.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US32787681A | 1981-12-07 | 1981-12-07 | |
| US327,876 | 1989-03-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1209038A true CA1209038A (en) | 1986-08-05 |
Family
ID=23278463
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000417034A Expired CA1209038A (en) | 1981-12-07 | 1982-12-06 | Crystalline human leukocyte interferon |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0083734B1 (en) |
| JP (1) | JPS58164597A (en) |
| AT (1) | ATE18410T1 (en) |
| AU (1) | AU551407B2 (en) |
| CA (1) | CA1209038A (en) |
| DE (1) | DE3269727D1 (en) |
| DK (1) | DK159164C (en) |
| IE (1) | IE54374B1 (en) |
| IL (1) | IL67411A0 (en) |
| NZ (1) | NZ202698A (en) |
| PH (1) | PH20413A (en) |
| ZA (1) | ZA828917B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5441734A (en) * | 1993-02-25 | 1995-08-15 | Schering Corporation | Metal-interferon-alpha crystals |
| CN1245215C (en) | 2001-02-28 | 2006-03-15 | 四川省生物工程研究中心 | Recombination high efficiency composite interferon used as hepatitis B surface antigen and e antigen inhibitor |
| US7585647B2 (en) | 2003-08-28 | 2009-09-08 | Guangwen Wei | Nucleic acid encoding recombinant interferon |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ZA796175B (en) * | 1978-11-24 | 1980-11-26 | Hoffmann La Roche | Purified proteins and process therefor |
-
1982
- 1982-12-03 DE DE8282111201T patent/DE3269727D1/en not_active Expired
- 1982-12-03 ZA ZA828917A patent/ZA828917B/en unknown
- 1982-12-03 DK DK538482A patent/DK159164C/en not_active IP Right Cessation
- 1982-12-03 NZ NZ202698A patent/NZ202698A/en unknown
- 1982-12-03 EP EP82111201A patent/EP0083734B1/en not_active Expired
- 1982-12-03 IL IL67411A patent/IL67411A0/en not_active IP Right Cessation
- 1982-12-03 AT AT82111201T patent/ATE18410T1/en not_active IP Right Cessation
- 1982-12-06 JP JP57213807A patent/JPS58164597A/en active Granted
- 1982-12-06 IE IE2882/82A patent/IE54374B1/en not_active IP Right Cessation
- 1982-12-06 AU AU91155/82A patent/AU551407B2/en not_active Ceased
- 1982-12-06 PH PH28226A patent/PH20413A/en unknown
- 1982-12-06 CA CA000417034A patent/CA1209038A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| IL67411A0 (en) | 1983-05-15 |
| ZA828917B (en) | 1983-09-28 |
| EP0083734A1 (en) | 1983-07-20 |
| DE3269727D1 (en) | 1986-04-10 |
| DK159164B (en) | 1990-09-10 |
| DK159164C (en) | 1991-02-11 |
| AU9115582A (en) | 1983-06-16 |
| IE54374B1 (en) | 1989-09-13 |
| PH20413A (en) | 1987-01-05 |
| NZ202698A (en) | 1986-02-21 |
| JPS58164597A (en) | 1983-09-29 |
| DK538482A (en) | 1983-06-08 |
| IE822882L (en) | 1983-06-07 |
| JPH0477000B2 (en) | 1992-12-07 |
| EP0083734B1 (en) | 1986-03-05 |
| AU551407B2 (en) | 1986-05-01 |
| ATE18410T1 (en) | 1986-03-15 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| MKEX | Expiry |