EP0027793A1 - Interferons d'origine animale et de grande purete - Google Patents

Interferons d'origine animale et de grande purete

Info

Publication number
EP0027793A1
EP0027793A1 EP19790901166 EP79901166A EP0027793A1 EP 0027793 A1 EP0027793 A1 EP 0027793A1 EP 19790901166 EP19790901166 EP 19790901166 EP 79901166 A EP79901166 A EP 79901166A EP 0027793 A1 EP0027793 A1 EP 0027793A1
Authority
EP
European Patent Office
Prior art keywords
interferon
leukocyte
human
porcine
bovine
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.)
Withdrawn
Application number
EP19790901166
Other languages
German (de)
English (en)
Inventor
William A. Carter
Frederick H. Johnson, Jr.
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hem Research Inc
Original Assignee
Hem Research Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Hem Research Inc filed Critical Hem Research Inc
Publication of EP0027793A1 publication Critical patent/EP0027793A1/fr
Withdrawn legal-status Critical Current

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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • bovine, ovine, equine, and porcine i.e., cattle, sheep, horses, and swine cross species lines, and are effective in human application.
  • the bovine and porcine interferon have tested out as the most effective, and are the preferred embodiments.
  • the high purity interferons seem essentially to be non-antigenic in the human.
  • the belief offered for the nonantigenity is that existence of several distinct forms of interferon in the human with differing antigenic determinants has created a wide enough zone of antigenic tolerance for differences in interferon moleculr structures such that interferons from bovine and porcine leukocytes, in particular, are tolerated by the human immune system and are pharmacologically quite potent in the human.
  • this invention relates to high purity interferons derived from porcine, equine, bovine and ovine. Since purity levels of proteins are difficult to establish, the term high purity as herein contemplated should be understood as the level of purity required to make the interferon essentially non-antigenic in humans. These high purity interferons are believed to be noval products. As a rule of thumb, high purity interferon comprises an interferon product with at least about
  • This invention also relates to the process of culturing leukocytes taken from the blood of porcine, bovine, ovine and equine in the presence of an interferon inducing agent, such as a virus for example, then recovering the interferon in high purity.
  • an interferon inducing agent such as a virus for example
  • the inducer is a virus virulent to the animal source of the leukocytes.
  • This invention also relates to a procedure for purifying crude interferon recovered from the culture medium to high purity levels, e.g. to more than 10 6 international reference units per mg of protein.
  • practice of this invention contemplates generation of interferon in a culture medium by appropriate challenge to porcine, bovine, equine and ovine leukocytes, followed by recovery and purification of the interferon.
  • the techniques developed for generating human leukocyte interferon e.g., as briefly described by Burke, supra
  • the leukocyte source materials herein contemplated offer many advantages to the interferon arts.
  • the output from a production facility is no longer restricted by the limited availability of human blood.
  • the leukocytes can be made available immediately after the blood is removed from the animal.
  • the plasma associated with the animal source leukocytes constitutes a by-product of possible value, being available for culture medium nutrient purposes, for example.
  • Fresh whole blood obtained by bleeding or butchering of the animals is separated (promptly) into its components, by conventional techniques, e.g., centrifugation to remove the plasma from cellular constituents, then the white cells or leukocytes are recovered e.g., as a buffy coat.
  • Commonly lysis is employed to eliminate a residual red cells contaminant from the recovered leukocytes. Lysis of red cells to separate them from leukocytes is also contemplated for practice of this invention.
  • the leukocytes from an animal source can be suspended in a nutrient medium and challenged by the interferon inducer, in as little as an hour after the whole blood is collected, and certainly while the leukocytes are still fresh and viable.
  • the nutrient medium in which the leukocytes are suspended and cultured may be derived in whole or in part from the plasma fraction of the whole blood.
  • the plasma contains a significant protein content of a nature which makes purification of the interferon product more difficult. (c.f. 3,800,035).
  • the exact proportion of plasma employed in the nutrient medium is a matter of coice to be weighed against relative costs of purifying the interferon generated therein to high purity levels.
  • the species divorcement between host animal and patient created by practice of this invention allows employment of a wider variety of viral inducers than heretofore possible, including notably the possibility offered for using a virus which attacks the host species.
  • the interferon inducer may be any of the many materials heretofore suggested to the art for inducing leukocytes to produce interferon, including viruses such as for example Newscastle disease virus, (NDV), Sendai virus, etc., and synthetic materials such as double stranded ribonucleic acids or single stranded ribonucleic acids, complexed or uncomplexed.
  • NDV Newscastle disease virus
  • Sendai virus Sendai virus
  • synthetic materials such as double stranded ribonucleic acids or single stranded ribonucleic acids, complexed or uncomplexed.
  • BTV blue tongue virus
  • a special leukocyte/ inducer interaction seems to take place.
  • the minimum number of affective BTV virus particles per leukocyte cell necessary to induce a high level of interferon in the culture medium was found to be half or less of the number for NDV.
  • Foot-and-mouth disease (FMDV) was also found to be an unusually efficient inducer of swine and bovine leukocyte interferon, requiring vis a vis NDV a lower multiplicity of infection (moi) or number of virus particles per leukocyte during the inital interferon infection phase.
  • the suspended leukocytes are optimally primed with homologous interferon 10-50 units per 10 7 leukocyte cell count for about 2 hours, then are challenged with a virus 1 to 10 x 10 8 plaque forming units per 10 7 cell count. After 6-48 hours of incubation the cells are removed from the medium, e.g., by centrifugation, then the medium is dialyzed overnight against saline of pH-2 (to deactivate the virus). Thereafter the interferon is harvested after readjustment of the pH to 6.5-7.0. The interferon is then purified by affinity adsorption techniques consisting primarily of hydrophobic and lectin chrpmatography as Biochemistry 1976, pages 5182-5187, Vol 251 No. 15; Journal of
  • the purification recovery sequence recovers about 15% of the initial interferon activity in the form of high purity interferon with more than about 10 international reference units per mg of protein.
  • the final solution contains more than one million biological units per ml of solution.
  • the concentration purification of interferon is in excess of 1000 fold.
  • the crude interferon is of about 100-1000 biological units/ml of medium, specific activity of 5x10 2 to 5x10 3 international reference units/ mg of protein (measured by Lowery or luorometric methods).
  • the purification sequence now described purifies the (porcine) leukocyte interferon several thousand fold, resulting in a highly purified non-antigenic product suitable for human use as a parenteral drug.
  • the crude product was passed through a column of concanavalin A, as described in Biochemistry, 1976, page 704 Vol 15. The majority of interferon passes through the column largely unretarded whereas contaminating glycoproteins having the prosthetic group: /
  • the leukocyte interferon solution was passed through a column of blue dextran (cibacron Blue F3GA-dextran) which had been immobilized on cyanogen bromide activated agarose as described, in Biochemistry, 1976, pages 5182-5187, Vol 15.
  • the chromatography principle here employed is hydrophobic chromatography and the results indicate that the interferon is a hydrophobic protein which can be separated effectively from other contamination proteins in porcine and bovine sera. It is specifically observed that the serum albumin, a potential antigenic protein for man, is efficiently removed at this step as it passes through the column essentially unretarded, whereas the interferon requires a high concentration of sodium chloride for its efficient elution.
  • the leukocyte interferon was bound to a straight chain hydrocarbon decyl (C 10 ) agarose column.
  • the interferon was bound tightly whereas most of the remaining contaminating protein were bound less tightly and could be removed with a solution of sodium phosphate, the interferon requiring an elutant consisting of 1M NaCl and a polarity reducing agent, ethylene glycol for its full and efficient removal.
  • the manner of purifying animal inter feron by hydrophobic chromatography is known to the art, e.g., see Journal of Biolgoical Chemistry, 1976, pages 7620-7625, Vol 251 no. 23.
  • the highly purified leukocyte interferon was further freed to high molecular weight and very low molecular weight contaminating proteins by the principle of molecular sieving using a Sephadex G-100 column precalibrated with known molecular weight markers such as bovine serum albumin, ovalbumin, soybean trypsin inhibitor, chymotrypsinogin and ribonuclease, etc. All interferon activity was eluted completely in the zone between ribonuclease and ovalbumin and particularly close to chymotrypsinogen, thus indicating its apparent molecular weight of about 20,000 to 30,000 daltons.
  • molecular weight markers such as bovine serum albumin, ovalbumin, soybean trypsin inhibitor, chymotrypsinogin and ribonuclease, etc. All interferon activity was eluted completely in the zone between ribonuclease and ovalbumin and particularly close to chymotrypsinogen, thus indicating its
  • Interferon was assayed in monolayers of several cell lines including porcine kidney, bovine skin and human fibroblasts.
  • the colorimetric method of Finter was used, see Journal of General Virology, 1969, pages 419-427, Vol 5, and Journal of Molecular Biology, 1972, pages 567-587, Vol 70.
  • Vesicular stomatitis virus at a multiplicity of infection of about 0.15 plaque forming unit/cell, was the challenge virus.
  • An international reference standard of human interferon was used also.
  • porcine leukocyte interferon A typical example of the assay illustrating that porcine (and bovine) leukocyte interferon are suitable for human use is as follows: aliquots of a solution containing a known quantity of 100 units of porcine leukocyte interferon was added to tissue culture wells (cell monolayers) of porcine, human and bovine cells. Forty to fifty percent of the activity observed in the homologous cell was observed in the human cells. That is, the 100 units of porcine leukocyte interferon assayed as 40 to 50 units in the heterologous cells of human fibroblasts. In normal (by karyology) human cells highly sensitive to the interferon effect the porcine leukocyte interferon was often 100 percent active in the human cells tested.
  • a human cell line which has a trisomy of the 21st chromosome was used for the test cell.
  • the 21st chromosome c.f. Burke, supra, is responsible for formation of specific receptors on the surface of the human cell for interferon.
  • the 100 units of porcine leukocyte interferon (as assayed on porcine cells) actually measured 300-500 units illustrating the high activity of this purified porcine leukocyte interferon for human tissue and its strong ability to protect these cells against cytolytic viral infections.
  • the activity indicated the existence of an extensive region of homology, or similarity, in amino acid sequence between porcine leukocyte interferon and human leukocyte interferon.
  • a precedence for such similarity is known elsewhere in the arts as in the example of the close similarity between porcine and bovine insulins, and human insulin.
  • a sample of 125 ml of whole blood and 30 ml ACD (a commercially available anticoagulant) was concentrated at 400 rpm for 10 minutes in 125 ml conical bottles. Then after differential centrifugation the fractions containing the plasma and white cells were added to a 50 ml syringe pre-loaded with 5.0 ml of plasmogel and 7.5 ml ACD, total volume being 50 ml. The syringe contents were mixed well, then the syringe was inverted and left to stand for 45 minutes. The upper fraction of the syringe contents was expressed into a 40 ml centrifugal tube and spun 500 rpm for 10 minutes.
  • the white cells were resuspended to 12.0 ml (of medium 199 or RPMI 1640) induced with 0.6 ml of NDV
  • the suspension was clarified at 10,000 rpm for 10 minutes.
  • the supernatant, pH 7.12 was acidified to pH 2.0 (with IN HCL), and left overnight, then dialyzed for 60 hours at 4°C against 0.15-M Na Cl, pH 2. (If desired the dialysis step may be omitted).
  • the dialysis solution was then, changed to a solution of phosphate buffered saline adjusted to pH 7.4 and the leukocyte interferon was dialyzed for an additional 24 hours.
  • Bovine and Porcine Leukocyte Separation Two 15 ml samples each of both calf and porcine whole blood were isolated and treated, each with 120 ml of cold 0.83% ammonium chloride and stood for 10 minutes on ice. The resulting lysates were centrifuged for 10 minutes at 400 rpm. This method, utilizing ammonium chloride lysis, greatly reduces the residual number of red blood cells.
  • the pooled pellets (2.0 ml) had a Coulter count of
  • Example 2B Purification of Bovine and Porcine Leukocyte Interferon of Example 2
  • samples of the PLIF and BLIF from column, 4 were chromatographed on phenyl Sepharose CL-4B. Samples were first dialyzed against 0.15M NaCl in 0.02 M sodium phosphate pH 7.4
  • PBS porcine interferon
  • the bovine interferon was about 5 x 10 7 international reference units per mg.
  • the additional purification of step 5 was 5-10 fold resulting in an overall purification of 10,000 to
  • Example 2 The same separating procedure as in Example 1 was applied to horse and ovine whole blood. In the instance of the horse blood the separation was quite good and the buffy coat could be clearly picked off, which made the total volume in the syringe only 40 ml.
  • RATIO inducer overall purifibiological recovery cation activity factor normal diploid over homo. cell equine leukocyte interferon (rl n rC n ) 45% 300 fold 0.05
  • ovine leukocyte interferon rl n rC n
  • the purification sequence does not appear as good for equine and ovine as for bovine and porcine interferons.
  • the unit activity of interferons are very high, at least 10 8 International Reference Units per mg of protein, and moreover that more than one interferon component is elaborated by human leukocytes in initially a very low concentration in the fluid.
  • the very high degree of concentration desired for clinical products e.g., from 10 3 units activity to about 10 6 unit activity, requires that the purification technique for human interferon products be extremely selective for what is recovered. Even minor chemical and/or conformal differences between the protein molecule of the interferon sought and impurity proteins, even if the impurity constituted a different interferon molecule will cause the impurity to be rejected by the purification sequence. Accordingly past efforts to concentrate human interferon a thousand-fold are likely to have involved complete loss of interferon components that differ chemically or conformally from the interferon component successfully concentrated.
  • the yield differences are 10-20%, sometimes even more, and occur both from the purification procedure described above and from adaptation of the Cantell procedure to the crude animal leukocyte interferon.
  • the yield differences are believed to indicate selectively complete loss of some interferon component or components, which components are believed to have lower or perhaps non-existent species cross-over potential.
  • C-IF crude concentrated interferon
  • P-IF partially-purified interferon, fraction B
  • PBS 0.01 M phosphate-buffered saline, pH 7.2
  • SDS sodium dodecyl sulphate
  • KSCN potassium thiocyanate
  • ppt precipitate
  • spn supernatant.
  • the specific activity of the animal leukocyte interferon to human cells was about 10 6 International Reference Units per mg of protein.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Interferons provenant de races bovine, porcine, chevaline et ovine, de haute purete efficace sur differentes lignes d'especes. Les interferons sont obtenus en provoquant ou amorcant des leucocytes viables in vitro avec un inducteur d'interferons, de preference un inducteur infectieux sur l'animal hote, puis en recuperant l'interferon du milieu de culture. Apres purification jusqu'a une valeur depassant 106 Unites de Reference Internationale par mg de proteine les interferons d'origine animale de haute purete sont non antigenes chez les humains.
EP19790901166 1979-04-27 1980-11-17 Interferons d'origine animale et de grande purete Withdrawn EP0027793A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1979/000268 WO1980002375A1 (fr) 1977-09-23 1979-04-27 Interferons d'origine animale et de grande purete

Publications (1)

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EP0027793A1 true EP0027793A1 (fr) 1981-05-06

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EP19790901166 Withdrawn EP0027793A1 (fr) 1979-04-27 1980-11-17 Interferons d'origine animale et de grande purete

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EP (1) EP0027793A1 (fr)
WO (1) WO1980002375A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IE54592B1 (en) * 1982-03-08 1989-12-06 Genentech Inc Anumal interferons, processes involved in their production, compositions containing them, dna sequences coding therefor and espression vehicles containing such sequences and cells transformed thereby
US5827694A (en) * 1982-03-08 1998-10-27 Genentech, Inc. DNA encoding non-human animal interferons, vectors and hosts therefor, and recombinant production of IFN polypeptides
US6432677B1 (en) 1982-03-08 2002-08-13 Genentech, Inc. Non-human animal interferons
JPS5925332A (ja) * 1982-08-04 1984-02-09 Mochida Pharmaceut Co Ltd 抗腫瘍性糖蛋白質の製法
US5831023A (en) * 1982-11-01 1998-11-03 Genentech, Inc. Recombinant animal interferon polypeptides
JPS60500574A (ja) * 1983-02-24 1985-04-25 インステイチユト　オルガニチエスコゴ　シンテザ　アカデミイ　ナウク　ラトビイスコイ　エスエスア−ル ヒト白血球インタ−フェロンｎ及び細菌細胞におけるその製造方法
IL71555A (en) * 1984-04-15 1992-06-21 State Of Israel Israel Inst Fo Bovine interferon
FI90990C (fi) * 1984-12-18 1994-04-25 Boehringer Ingelheim Int Rekombinantti-DNA-molekyyli, transformoitu isäntäorganismi ja menetelmä interferonin valmistamiseksi
US4689224A (en) * 1985-10-07 1987-08-25 Neogen Corporation Method for administering vaccines containing equine leukokines and compositions therefor
WO1989009065A1 (fr) * 1988-03-23 1989-10-05 University Of Georgia Research Foundation, Inc. Production a grande echelle d'interferon de leucocytes bovins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO8002375A1 *

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Publication number Publication date
WO1980002375A1 (fr) 1980-11-13

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