EP1140144A2 - Procedes et composition de melanges naturels extremement purifies d'interferons de type i derives des leucocytes - Google Patents

Procedes et composition de melanges naturels extremement purifies d'interferons de type i derives des leucocytes

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Publication number
EP1140144A2
EP1140144A2 EP99967753A EP99967753A EP1140144A2 EP 1140144 A2 EP1140144 A2 EP 1140144A2 EP 99967753 A EP99967753 A EP 99967753A EP 99967753 A EP99967753 A EP 99967753A EP 1140144 A2 EP1140144 A2 EP 1140144A2
Authority
EP
European Patent Office
Prior art keywords
ifn
interferon
alpha
mixture
type
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Withdrawn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
EP99967753A
Other languages
German (de)
English (en)
Other versions
EP1140144A4 (fr
Inventor
Joseph P. Morris
Duy Nguyen
James Kappelman
Mead M. Mccabe
Michael D. Potter
M. Reza Ziai
Stephen Feldman
Hipolito Hartman
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.)
Viragen Inc
Original Assignee
Viragen Inc
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Filing date
Publication date
Priority claimed from US09/224,895 external-priority patent/US6350589B1/en
Priority claimed from US09/229,039 external-priority patent/US6433144B1/en
Application filed by Viragen Inc filed Critical Viragen Inc
Publication of EP1140144A2 publication Critical patent/EP1140144A2/fr
Publication of EP1140144A4 publication Critical patent/EP1140144A4/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Withdrawn legal-status Critical Current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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to methods for isolating highly-purified mixtures of natural type I interferons from white blood cells, and particularly human white blood cells.
  • the invention also 5 relates to highly-purified mixtures of natural type I interferons which resemble natural type I interferon in that it includes 9 subtypes, i.e., alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha- 14, alpha-21 and omega, giving rise to possibly 20 molecular species, including alpha-la, alpha-lnew, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha- 10a, alpha- 14a, alpha 14-b, alpha 14-c, alpha- 14LG, alpha-2 la, alpha-2 lb, 0 alpha-21 c, omega and omega LG.
  • the interferons are a family of proinflammatory cytokines important in mediating nonspecific host defense. While of critical importance in initiating anti-viral immunity, the family also acts as a potent initiator of cell growth and differentiation.
  • Type I interferon is a 5 designation for a family of related interferons that can include multiple subtypes of alpha interferon, beta interferon, omega interferons, and in some species the related trophoblast tau interferon.
  • the proteins are structurally similar, share common receptors, have common biological, activities and may share a common genetic locus.
  • the type I interferons are believed to have three major functions. First, they inhibit viral replication by activating cellular genes 0 that inhibit protein synthesis, thus contributing to the suppression of viral replication.
  • interferon preparations including leukemias (US 5,830,455), basal cell carcinomas (US 5,028,422), squamous cell carcinomas (5,256,410), breast cancer (US 5,024,833), gastrointestinal malignancies (US 5,444,064; 5,814,640), actinic keratoses (US 5,002,764), as well as macular degeneration (US 5,632,984), autoimmune disorders (5,830,456), diabetes (WO09806431A2), bacterial infections (US 5,817,307), and viral infections (US 5,830,456), including genital warts (US 4,959,210), hepatitis B (WO09823285A1), and herpes zoster and psoriasis (US 4,957,734). While the pharmaceutical applications of this family of cytokines is only beginning to be understood, the problems related to
  • Natural interferon production has traditionally involved ammonium chloride treatment of buffy coats to lyse the red blood cells and to isolate the leukocytes, followed by viral stimulation of leukocytes with subsequent large scale harvesting of culture medium. The interferons are then isolated by various precipitation, adsorption, or immuno-aff ⁇ nity techniques.
  • the prior art usually employs serum in the culture of leukocytes, which significantly contributes to the resulting contamination of the secreted protein product.
  • the use of viral preparations to induce interferon production also adds a significant source of contaminating material.
  • the prior art has heretofore not addressed these problems.
  • U.S. Patent No. 5,503,828 describes an alpha-interferon composition characterized by having at least 50% of alleles of ⁇ 2 and ⁇ 8, and one or more additional alpha interferon species selected from the group consisting of ⁇ 4, ⁇ 7, otlO, ⁇ l6, ⁇ l7, and ⁇ 21. While U.S. Patent No. 4,503,035 teaches a preparation of certain interferon species, the preparation does not include for example, alpha-1, alpha-5, alpha-14 and omega subtypes. Thus, a natural mixture of highly pure interferon having a full spectrum of subtypes is not taught by this U.S. Patent No. 4,503,035.
  • U.S. Patent No. 5,762,923 teaches an aqueous interferon composition dissolved in water with a non-ionic detergent and benzyl alcohol in amounts sufficient to stabilize the interferon-alpha.
  • the composition also contains an acidic buffer which provides a pH of 4.5 to 6.0, and may also contain an isotonizing agent.
  • U.S. Patent No. 4,847,079 teaches a stable pharmaceutical composition of interferon and thimerosal which is resistant to microorganism contamination and growth.
  • 4,675,184 teaches a stabilized interferon with 15 to 60% by weight of a tri or higher polyhydric sugar alcohol and an organic acid buffer as stabilizers, and a conventional pharmaceutical carrier or diluent at pH about 3 to 6.
  • the composition can further contain an anionic surfactant and/or albumin as a stabilizer.
  • U.S. Patent No. 5,236,707 teaches the use of amine stabilizing agents such as primary aliphatic amines and anionic stabilizing agents such as lithium organo sulfates which protect human interferons from degradation and provide enhanced storage stability.
  • U.S. Patent No. 5,431,909 teaches the use of amine stabilizing agents such as primary aliphatic amines and anionic stabilizing agents such as lithium organo sulfates to protect human interferons from degradation and provide enhanced storage stability.
  • U.S. Patent No. 4,780,413 relates to the production of interferon by adding an inducer to lymphoblastoid cells.
  • U.S. Patent No. 4,172,071 relates to a process for the purification of interferon by absorption onto chromophore blue columns and elution with a low salt buffer.
  • U.S. Patent No. 4,289,689 combines affinity chromatography with high pressure liquid chromatography to purify interferons from fibroblasts.
  • U.S. Patent No. 4,465,622 describes a method of adsorbing interferon onto a carrier containing acrylonitrile polymer and eluting the adsorbed interferon with an appropriate buffer.
  • 4,485,017 discloses a process wherein a partially purified preparation is passed through an antibody affinity column and a reversed-phase high performance liquid chromatographic column. Organic solvents used during the elution are extracted and the protein concentrated for subsequent use.
  • U.S. Patent No. 4,551,271 describes the purification of solutions of recombinant interferons by chromatography on metal chelate resins, including copper or nickel.
  • U.S. Patent No. 5,391,713 describes a process for purification of human leukocyte interferon which includes immunoaffinity chromatography, ion-exchange chromatography, and a series of precipitation and centrifugation steps.
  • the present invention alleviates and overcomes certain of the above-identified problems and shortcomings of the present state of interferons through the discovery of novel highly purified mixtures of Type I interferon derived from white blood cells, and novel methods of isolating and using same.
  • the multisubtype Type I interferons of the present invention are a highly purified blend of natural ⁇ and ⁇ interferons obtained from leukocytes.
  • the leukocytes may be derived from blood or a blood component, such as an apherisis product. It is believed that no proteins, other than interferon proteins, can be detected in the multisubtype Type I interferons of the present invention using standard gel elctrophoresis techniques.
  • the multisubtype Type I interferons of the present invention do not contain any nucleic acids. It is believed that the multisubtype Type I interferons of the present invention achieve a purity of at least about 95%, and up to about 98%.
  • the multisubtype Type I interferons of the present invention are virtually free of contaminants, such as serum albumin and other low and high molecular weight proteins.
  • the molecular weights of the multisubtype Type I interferons of the present invention are generally between about 10,000 and about 30,000 Daltons, and more particularly between about 19,000 and 27,000 Daltons, as measured by SDS-Page.
  • the multisubtype Type I interferons of the present invention have an activity of at least about 1 x 10 8 units, as measured by a standard anti-viral assay conatining an international interferon standard, and apparent isoelectric points of between about 5.0 and about 8.5.
  • the multisubtype Type I interferons of the present invention include both naturally glycosylated and naturally unglycosylated forms of interferon subtypes.
  • the naturally glycosylated subtypes are believed to include alpha-2 species, alpha-14 species, alpha-2 la and omega.
  • the multisubtype Type I interferons of the present invention resembles natural Type I interferon and in particular, natural human Type I interferon in that it contains a mixture of multiple INF- ⁇ and INF- ⁇ subtypes derived from white blood cells. More specifically, there are believed to be at least 9 different subtypes that can give rise to at least 16, and possibly 19 or more molecular species.
  • the 9 subtypes include alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 and omega, whereas the 19 molecular species include alpha- la, alpha-lnew, alpha-2a, alpha-2b, alpba-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha- 10a, alpha- 14a, alphal4-b, alpha 14-c, alpha- 14LG, alpha-2 la, alpha-2 lb, omega and omega LG.
  • the multisubtype Type I interferons of the present invention contain a plurality or a significant number of different Type I interferon subtypes, it is believed that the multisubtype Type I interferons of the present invention very closely resemble the natural Type I interferon system and, in particular, the natural human Type I interferon system produced by and operating within humans, especially when compared to the recombinant monocomponent interferon products available heretofore.
  • the present invention also contemplates novel procedures for obtaining the multisubtype Type I interferons.
  • the present invention is concerned with methods of obtaining a highly purified mixture of Type I interfeon having a plurality of subtypes from leukocytes wherein the highly purified mixture of Type I interferon has a purity of at least about 95%, contains substantially only interferon proteins, contains at least 9 different subtypes, including alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 and omega which gives rise to at least 16 and possibly 19 or more molecular species, and contains no more than about 35% by weight IFN ⁇ -2 and IFN ⁇ -8 subtypes.
  • Such a method comprises: (a) culturing leukocytes; (b) stimulating the leukocytes to produce a crude interferon; (c) concentrating the crude interferon to remove low-molecular weight contaminants; (d) liquid volume to produce a concentrated crude interferon; (e) removing a substantial amount of serum albumin and other contaminants from the concentrated crude interferon to produce a partially purified interferon mixture containing a plurality of subtypes; (f) removing substantially all remaining serum albumin and other contaminants from the partially purified interferon mixture to generate an interferon mixture having a purity of between about 50% and about 80%; and (g) purifying the about 50% to about 80% interferon mixture to produce a highly purified mixture of Type I interferon having a purity of at least about 95% and containing no more than about 35% by weight IFN ⁇ -2 and IFN ⁇ -8 subtypes.
  • Such a method may include the further step of isolating the leukocytes from blood or a blood component,
  • the acquisition of leukocyte segment results in an increase in the number of leukocytes due to better component manufacturing procedures.
  • the leukocytes are generally more hardy and have increased productivity due to the handling and isolation procedures.
  • there is improved productivity of a "unit" of leukocytes due to improved recovery from each donation, increased productivity due to additives added at the "buffy coat” stage and temperature maintenance procedures.
  • the additives into the buffy coat are believed to improve white blood cell health and separation.
  • the procedure of the present invention is time sensitive from the nature of the donation to component manufacture to the PMBC isolation segment to IFN production.
  • the procedures of the present invention requires the use of gas permeable bags during storage of the harvested white blood cells and temperature maintenance (ambient, RT) during all steps leading from 1 donation to culture.
  • the PMBCs are generally healthier because the procedures of the present invention gently remove red blood cells from the white blood cells without lysis, the PMBCs are maintained at physiological ionic strength throughout the PBMC isolation process, the white blood cells are gently washed, and the PMBCs are gently isolated via use of LSM at manufacturing scale. Moreover, because plasma and plasma proteins are removed, the use of non-immunoaffinity purification procedures can be used downstream to attain a purity of greater than about 95%. Finally, because the PMBCs are isolated, this limits proteolysis in cell culture.
  • PMNs plymorphonuclear leukocytes
  • use of purified Sendai Virus in cell culture and protein free medium enhances purification, limits the need for immunoaffinity, and removes any non-human proteins from the system.
  • protease inhibitors should be used during cell culture and suicide inhibitors of protease activity should be used immediately after cell culture to prevent product proteolysis.
  • pH, temperature, p ⁇ 2, prevention of PMN breakdown, and cell health should be controlled during cell culture.
  • monocytes in the cell culture and presence of an inducing factor during the first two hours of cell culture after Sendai addition are generally needed.
  • the protein burden of all steps leading to purification is reduced so that non- immunoaffinity and non-RP-HPLC methods can be effectively employed for purification.
  • This benefit reduces costs, limits structure degradation and enhances the full recovery of Type I interferon subtypes.
  • a reduction in protein burden suring this segment is accomplished by using protein free cell culture medium, purified Sendai, extensive washing of isolated white blood cells and removal of PMNs to prevent the release of their cellular contents into the purification stream.
  • this benefit provides for the capture of a multisubtype Type I interferon which contains a mixture of multiple INF- ⁇ and INF- ⁇ subtypes derived from white blood cells. This is believed to be particularly advantageous in view of the strong anti-viral activity noted for INF- ⁇ subtypes.
  • BC - Buffy coat BCA - Assay used for protein concentration determination. Capture pool - concentrated interferon prepared from a culture medium by, for example, Sepharose Big Bead chromatography.
  • Natural Mixture of Type I IFN or Interferon - Natural mixture of type I INF or interferon, or any similar phrase, refers to any natural type I interferon obtained from white blood cells which comprises a blend of nine sub-types, i.e., ⁇ l, ⁇ .2, ⁇ 5, ⁇ 7, ⁇ 8, ⁇ lO, l4, ⁇ 21, and ⁇ ), giving rise to at least 16, and possibly 20 or more, different molecular species, including alpha- la, alpha-lnew, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha- 10a, alpha- 14a, alpha 14-b, alpha 14-c, alpha-14LG, alpha-21a, alpha-21b, alpha-21c, omega, omega LG and/or others.
  • ⁇ l, ⁇ .2, ⁇ 5, ⁇ 7, ⁇ 8, ⁇ lO, l4, ⁇ 21, and ⁇
  • LSMTM Lymphocyte Separation Medium. Contains ficol and hypaque.
  • MES 2-(N-Morpholino)ethanesulphonic acid.
  • PBS phosphate buffered saline.
  • PBMC peripheral blood mononuclear cells.
  • PefablocTM A commercially available serine protease inhibitor or its equivalent. Also, Pefabloc SCTM.
  • PercollTM A commercially available solution of low osmolarity ( ⁇ 25 mOsm/kg H 2 0) with variously sized silica particles. Any equivalent may be used PF68-Pluronic acid F-68.
  • RB-1 refined buffy coat. Prepared by washing crude buffy coats with PBS (see example 2).
  • RP-HPLC Reversed phase high performance liquid chromatography.
  • RPM1 Nutrient medium that supports leukocyte culture.
  • Semi-permeable bag Also semi-permeable container. Any container with a large surface area to volume ratio that allows gas to pass through the container walls.
  • Serum-free culture medium any culture medium that can be used to support the growth of leukocytes and their production of interferon, but that does not contain any fetal calf or other type of serum. This includes at least RPMI, and MEM as well as others known to those of skill in the art.
  • Tris - ⁇ -Tris(hydroxymethyl)aminomethane VSV - vesicular stomatitis virus. Used in a bioassay to assess interferon anti-viral activity.
  • One aspect of the current invention concerns a highly purified mixture of Type I interferons containing at least 9 subtypes which gives rise to at least 16, and possibly up to 20 or more, molecular species.
  • the 9 subtypes include IFN- ⁇ l, IFN- ⁇ 2, IFN- 5, IFN- ⁇ 7, IFN- ⁇ 8, IFN- ⁇ lO, IFN- ⁇ l4, IFN- ⁇ 21 and IFN- ⁇ .
  • the molecular species include IFN- ⁇ la, IFN- ⁇ lnew, IFN- ⁇ 2a, IFN ⁇ 2b and/or IFN- ⁇ 2c, IFN- ⁇ 5, IFN- ⁇ 5LG, IFN- ⁇ 7, IFN- ⁇ 8a, IFN- ⁇ 8c, IFN- ⁇ lOa, IFN- ⁇ l4a and/or IFN- ⁇ l4b and/or IFN- l4c, IFN- ⁇ l4LG, IFN-21a and/or N- ⁇ 21b, IFN- ⁇ 21c, IFN- ⁇ and IFN- ⁇ LG.
  • natural mixture refers to the fact that the interferons are "native” or “natural”, e.g., not recombinant, and that they are purified as a mixture from white blood cells, rather than as individual subspecies which are then recombined.
  • IFN- ⁇ 7, IFN- ⁇ 8, IFN- ⁇ lO, IFN- ⁇ l4 include the additional subtypes IFN- ⁇ 7a, IFN- ⁇ 7b, IFN- ⁇ 7c, IFN- ⁇ 8b, IFN- ⁇ lOb. Additionally, subtypes IFN- ⁇ 4a, IFN- 4b, IFN- ⁇ l6, IFN- ⁇ l7a, IFN- ⁇ l7b, IFN- ⁇ l7c, and IFN- ⁇ l7d may also be present as suggested by preliminary experiments.
  • the major subtypes are about 25% IFN- ⁇ l (a and new), about, 15% IFN- ⁇ 2 (a and b and/or c), about 5% N-cc5 (a and LG), about 5% IFN- ⁇ 7, about 10% IFN- ⁇ 8 (a and c), about 10% IFN- ⁇ lOa, about 10% IFN- ⁇ l4 (a, b and/or c), about 10% IFN- ⁇ 21 (a, b and/or c) and about 5% IFN- ⁇ .
  • Type I interferon The highly purified natural mixtures of Type I interferon are stabilized in a buffer with the addition of about 1 mg/ml HSA. Although most prior art interferon solutions employ acidic buffers in which to formulate the interferon, it has been found that a neutral pH works best under the conditions described. It should also be understood that the natural mixtures of Type I interferon of the present invention are suitable candidates for standard freeze-drying techniques.
  • the interferon is kept in silanized vials at 4°C and the vials may be sparged with N2, if desired.
  • Any biocompatible buffer can be used to formulate the interferon and additional excipients and/or active ingredients may be added as necessary for the use and/or mode of application.
  • Type I interferon of the present invention contain a full spectrum of interferon subtypes which are substantially free of contaminating proteins thereby closely resembling the natural interferons produced by humans from leukocytes, they are particularly applicable to therapeutic uses.
  • the present invention contemplates a method of treating interferon-responsive diseases by administering an effective amount of a highly purified natural mixture of Type I interferon (IFN) isolated from white blood cells in accordance with the present invention in a pharmaceutically acceptable carrier, said natural mixture of type I IFN being at least about 95% pure before being combined with said pharmaceutically acceptable carrier, and said natural mixture of type I IFN comprising at least 9 subtypes which gives rise to at least 16, and possibly up to 20 or more, molecular species.
  • the 9 subtypes include IFN- ⁇ l, IFN- ⁇ 2, IFN-c-5, IFN- ⁇ 7, IFN- ⁇ 8, IFN- ⁇ lO, IFN- l4, IFN- ⁇ 21 and IFN- ⁇ .
  • the molecular species include IFN- ⁇ la, IFN- ⁇ lnew, IFN- ⁇ 2a, IFN- ⁇ 2b and/or IFN- ⁇ 2c, IFN- ⁇ 5, IFN- ⁇ 5LG, IFN- ⁇ 7, IFN- ⁇ 8a, IFN- ⁇ 8c, IFN- ⁇ lOa, IFN- ⁇ l4a, and/or IFN- ⁇ l4b and/or IFN- ⁇ l4c, IFN- ⁇ l4LG, IFN- ⁇ 21a and/or IFN- ⁇ 21b, IFN- ⁇ 21c, IFN- ⁇ and IFN- ⁇ LG.
  • the methods of the present invention concern those diseases or indications that are interferon-responsive and include, for example, hepatitis infection, such as hepatitis A infection, hepatitis B infection, hepatitis C infection, HIV infection, herpes zoster virus infection; influenza infection, common cold infections, hemorrhagic fever infections, genital warts, bacterial infections, chlamydia infection, Behcet's disease, Churg-Strauss syndrome, leukemia, T-cell leukemia, hairy cell leukemia, chronic myeloid leukemia, melanoma, myofibromatosis, T-cell lymphoma, basal cell carcinomas, squamous cell carcinomas, renal cell carcinoma, colorectal carcinoma, non-small cell lung cancer, cervical cancer, breast cancer, gastrointestinal malignancies, actinic keratoses, macular degeneration, autoimmune disorders, diabetes, psoriasis, multiple sclerosis, inflammatory bowel disease,
  • the highly purified natural mixtures of type I interferon (IFN), when intimately admixed in a pharmaceutical acceptable carrier, may be administered topically, orally, parenterally, sublingually, buccally, by nasal inhalation, rectally, vaginally, aurally, or ocularly.
  • IFN type I interferon
  • leukocytes begins with collecting, transporting and separating leukocytes from other blood cell fractions.
  • leukocytes have been collected as whole blood and stored in impermeable plastic bags at about 4 C to about 25 C temperature until processed into plasma and red blood cells.
  • the white blood cell layer (buffy coat) is generally a discarded side product of the process which can be collected and then treated with ammonium chloride to lyse the contaminating red blood cells.
  • the remained leukocytes are then cultured in media containing a serum such as fetal calf serum and activated with a viral inducer to produce interferon.
  • the transport of blood or buffy coats should be done in such a way as to minimize temperature variations, time of transport and maximize the oxygenation of the cells.
  • the use of semi-permeable plastic bags hung from the top of an insulating container so as to allow free flow of oxygen between and into the bags greatly increases the viability of the leukocytes. Further, the temperature should be maintained at 22° ⁇ 3°C. This has been achieved with the use of water bags inside the insulating container. Of course, the time between collection and processing should be minimized.
  • the various cells in the buffy coat are separated by density centrifugation on an LSMTM gradient with PercolF M overlay with a slightly mixed LSMTM/PercollTM interface.
  • the second cell layer, containing the PBMC is recovered with a peristaltic pump, washed in PBS and then transferred RPMI medium.
  • the use of this procedure minimizes the number of granulocytes, thus reducing the protease contaminant of later cultures. Throughout this procedure, it is important that the cells be maintained at a constant room temperature. Even a brief exposure to low temperatures can cause inadequate separation and lowered IFN production.
  • the PBMC cells are cultured in a serum-free medium, such as RPMI, in order to minimize contaminants throughout the later product purification procedures.
  • a serum-free medium such as RPMI
  • the cells are cultured with interferon primer at 37°C for about 2 hours.
  • Sendai virus is added and the culture continued for another 2 hours. Then the temperature is smoothly and quickly dropped to 28°C and culture continued for about 14 hours.
  • a protease inhibitor can be added, but this step is optional.
  • the Sendai virus that is used to stimulate interferon production is generally grown in the allantoic fluid of chicken eggs. This provides an additional source of contaminating proteins in the final interferon product. Therefore, in order to maximize product yield and purity, this additional source of contaminants is purified about 1000 fold by centrifugation of the Sendai virus on a potassium tartrate density gradient or by other methods known in the art.
  • the contaminating virus can be killed by incubation of the product at pH 2. Virus hemagglutination activity is eliminated in 30 minutes of low pH treatment. Further, interferon activity, as measured by anti-viral bioassay, is not negatively affected by up to 24 hours of acid treatment. This interferon capture pool is the starting product for the purification procedures discussed hereinafter.
  • the supernatant is removed from the cells and initially concentrated 100 fold by cation exchange chromatography.
  • concentration commonly practiced in the art are envisioned by the present invention, such as salt precipitation, ultrafiltration, dialysis, gel filtration, affinity chromatography, electrofocusing or electrophoresis or a combination of two or more of the above techniques.
  • the concentrated interferon obtained from leukocyte culture is then purified in three steps as follows.
  • interferon is isolated from the concentrate by hydroxyapatite (HA) chromatography at a low pH in the range of about 4.9 to about 5.2, and more preferably at about pH 5.0. This removes about 98% of the major contaminant which is human serum albumin, and other contaminating proteins, and provides an approximately 10 fold purification and additional concentration.
  • the partially purified interferon can be further purified by size exclusion chromatography (SEC) to remove the remaining human serum albumin and other minor contaminants. If desired, this is followed by anion exchange (AX) chromatography or hydrophobic interaction chromatography (HIC) or both.
  • SEC size exclusion chromatography
  • AX anion exchange
  • HIC hydrophobic interaction chromatography
  • a natural interferon mixture is produced that is between about 95% and 98% pure, and has the following characteristics: (a) it contains at least nine interferon subtypes giving rise to at least 16, and possibly 19 or more, molecular species, (b) a mixture of apparent molecular weights of between about 10,000 and about 30,000 Daltons and more particularly between about 19,000 and 27,000 Daltons, as measured by SDS-PAGE, (c) an activity of at least about or greater than 1 x 10 8 units, as measured by a standard anti-viral assay containing an international interferon standard, and (d) apparent isoeletric points of between about 5.0 and about 8.5.
  • the natural mixture of type I INF or interferon is thought to include both naturally glycosylated and naturally unglcosylated forms of interferon subtypes. Most subtypes, however, are thought to be unglycosylated.
  • the glycosylated subtypes are believed to include alpha -2 species, alpha-14 species, and omega species.
  • alpha- la alpha-lnew about 30% (la and lnew)
  • alpha-2a alpha-2b about 15% (2a and 2b and/or 2c) alpha-2c
  • alpha-8a alpha-8c about 10% (8a and 8c)
  • alpha- 14a alpha- 14b alpha- 14c alpha- 14LG about 10% (14a, 14b, 14c and/or 14LG)
  • alpha-2 la alpha-2 lb about, 10% (21a, 21b and or 21c) alpha-2 lc
  • the natural mixture of type INF or interferon may contain cytokine IL-6 in an amount of about 1/7000 to 1/500 of a clinically relevant therapeutic dose of IL-6.
  • interferon preparations such as WellferonTM (Burroughs-Wellcome), Alferon-n3TM (Interferon Sciences, Inc.), Roferon-2aTM (Roche Laboratories) and Intron-2bTM (Schering-Plough) reveals that these commercial preparations, in contrast, completely lack subtype-omega (as assayed by ELISA (Bonder Wein)). Moreover, because these interferon preparations do not contain a full spectrum of subtypes derived directly from white blood cells, it is believed that they cannot resemble the natural type I interferon produced by leukocytes within the body.
  • IL-6 is removed.
  • the amounts of the subtypes omega, alpha-14 and alpha-21 in the natural mixture of type I INF or interferon are substantially reduced. If the subtype omega is removed, the isoeletric point range changes from between about 5.0 to about 8.5 to between about 4.0 to about 6.0
  • the relative ratios of the subtypes in the natural mixture of type I INF or interferon produced by this method are adjusted appropriately by the removal of significant amounts of subtypes omega, alpha-14 and alpha-21.
  • ⁇ blood is collected at various collection centers and centrifuged to provide plasma, red blood cells and a buffy coat interface which can be separated and transported to a manufacturing facility.
  • Buffy coat viability is maximized by avoiding a high osmolarity environment and this can be achieved by washed with isotonic PBS or adding an appropriate stabilizer.
  • Buffy coats are transported in an insulating container (such as a Styrofoam cooler) of appropriate size for the number of bags to be transported.
  • the container is also packed with two bags containing water (or other fluid) at a temperature of 22° ⁇ 3°C in order to maintain the temperature inside the cooler at 22° ⁇ 3°C. Transport under these conditions provides reproducible interferon titers.
  • buffy coats or whole blood, leukocytes, or other cell fraction
  • a semi-permeable (gas permeable) bag or other container
  • hung inside the cooler in such a way as to ensure gas flow between the bags.
  • This can be done in a variety of ways. For example, by hanging the bags from a rack, clipping the bags to a rack or by equipping the bags themselves with a hanging means such as a hook (or eyehole) which can be set into an eyehole (or hook) on the lid, or the bag may have a rigid bar on the top edge which can be set into grooves in the cooler. Additional oxygenation can be achieved by sparging the container itself with O2 before transport or using bags with greater O2 permeability.
  • Density purification of while blood cells with ficoll-hypaque gradients is widely used in both research and clinical settings.
  • This technique combines an osmotic agent (hypaque) which dehydrates the red blood cells as they pass through the material.
  • a density gradient is formed by the dilution of the ficoll by the water displaced by the red blood cells and present in the plasma.
  • this method also causes the red blood cells to aggregate, thus further increasing their apparent density.
  • the commercial preparation LSMTM from Organon-Technica was selected as the reference method for these studies.
  • the PercollTM overlay is thought to work by retarding the entry of the PBMC into the LSMTM.
  • the lower resultant granulocyte concentration has the added benefit of reducing the protease content of the cultures, and thereby increasing the interferon yield.
  • the separation may fail entirely.
  • wash solutions such as HEPES or RPMI
  • the best results are obtained using PBS for all cell washes, prior to and following the PercollTM/LSMTM separation.
  • the three PBS washes (one during the RB-1 formation and two after harvesting the principal layer) effectively reduce the osmolarity to approximately 288 ⁇ 2 mOsm. It is discovered that monocyte survival is imperative for maximal natural mixture of type I INF or interferon production and that in contrast, granulocyte degradation leads to the degradation of the interferon. With the techniques described herein, the proportion of monocytes present in the final cell concentrate was consistently over 10%, and generally over 15%. The proportion of granulocytes was consistently under 20%, and generally under 10% and most frequently below 5%. Specifically, the procedure is as follows: The buffy coats are combined through a manifold via a peristaltic pump through thick silicon tubing (I.D. 5/16" O.D.
  • a specially designed manifold has been designed as to permit optimal cell collection rate and ease.
  • the peristaltic pump with manifold is gentler on the cells than a vacuum pump and provides better interferon titers. Care is taken not to introduce air into the system and minimize foaming, which is detrimental to the cells. Clamping the tubing between the manifold and pump while attaching and removing the leukopacs minimizes the introduced air.
  • the supernatant plasma is removed by gentle aspiration and a peristaltic pump.
  • Two aspirators are used for this process. One is bent at 90 degree and has a sealed tip and a slot in the upper surface of the short tube and is useful for separating the layers of supernatants. The tube is lowered just into the lower layer while the upper layer is aspirated. The second aspirator is also bent at a 90 degree angle and has a slightly flattened open tip which is useful for removing cells that adhere to the wall of the container.
  • the tubes are made with polycarbonate tubing which allows them to be autoclaved without breakage, although other materials may be used. Placing the bottles on a turntable may aid in successful aspiration.
  • the plasma volume that is removed is replaced with isotonic PBS (290 mOsm) and gently mixed by swirling the bottle.
  • the cells are concentrated by centrifugation for 15 minutes at 800 G (1700 rpm). This forms a refined buffy coat (RB-1) which is collected via peristaltica spiration into a separate flask.
  • the second PBMC containing layer is collected, diluted at least five fold in PBS and centrifuged for 10 minutes at 500 G (1345 rpm).
  • the cell pellet is resuspended in PBS in the original volume and the centrifuge repeated.
  • the cells are then suspended in RPMI medium and combined in a two liter bottle. The bottle is placed on an orbital shaker until diluted and used in cell culture.
  • EXAMPLE 3. CULTURE OF PERIPHERAL BLOOD MONONUCLEAR CELLS
  • the cell concentrate from Example 2 is diluted to approximately 107 cells/ml in RPMI medium.
  • the RPMI is supplemented with 5.958 g/1 HEPES, 2 g/1 NaHCO3, and 0.05% PF-68.
  • the temperature of the water bath is dropped smoothly and slowly to 28°C (this is done by adding ice over a period of 15 minutes) and the cells cultured for about 14 more hours. At the end of this period, the medium is harvested.
  • the protease inhibitor PefablocTM or other similar protease inhibitors can be added 30 minutes before the end of culture at a final concentration of about 0.5 to about 5 mM.
  • PefablocTM is kept as a 100X frozen aliquot at -70°C.
  • a variety of suicide protease inhibitors have been tested for their ability to function in this regard, but PefablocTM provides almost complete inhibition and can be removed by size exclusion chromatography.
  • a decoy substrate such as the peptide ala-ala-ala may be employed throughout the cell culture process.
  • this protocol uses Sendai virus that is purchased from SPAFAS, Inc. Storres, Connecticut which provides batches uniform in hemagglutination and interferon stimulatory activity. Because the virus is grown in the allantoic fluid of chicken eggs, the inducing virus contributes contaminating proteins to the culture which can later interfere with the purification of interferon. Therefore, it is preferred that the virus be purified by centrifugation on a 10% to 50% (w/w) density gradient of potassium tartrate before use. After centrifugation, the viral fractions are isolated, concentrated and resuspended in PBS buffer. Other methods of purification are envisioned in accordance with techniques known in the art.
  • the protocol requires thawing frozen virus at 37°C with constant stirring.
  • a clarifying centrifuge is performed to remove the feathers and red blood cells (Beckman J2-21 centrifuge with JCF-Z rotor). The clarified pool is held overnight at 40°C. (((Why?)))
  • a 10- 50% potassium tartrate gradient is established in Electronucleonics Model K centrifuge with Super G rotor. The clarified virus applied to the gradient and centrifuged for 18 hours at 100,000 G (a Beckman L8-80 ultracentrifuge with SW-28 swinging bucket rotor can be used for small preps). The gradient is eluted from the bottom of the tube and fractionated. The early peak (about 3/4 into the gradient) contains the viral particles. The particles are concentrated by centrifugation and resuspended in 1/lOOth the original crude volume with PBS. EXAMPLE 5.
  • VIRUS INACTIVATION Culture media (CM, from example 3) is collected and the pH is dropped to pH 2.75
  • the acid should be added at a rate of about 5-20 ml min with continuous mixing of the harvested CM.
  • the acidified solution is stored at about 4°C for 2 to 4 hours. Viral hemagglutination activity is eliminated in about thirty minutes or less with no detectable loss in interferon activity as measured by VSV assay.
  • the pH of the CM is brought to about 4.40 (range 4.3 to 4.5) and the CM is prefiltered using a 0.45 ⁇ m filter (Millipak 200 Filter Unit (P/N: MPHL 20C A3)), followed by filtration with a 0.22 ⁇ m filter (Millipak 60 Filter Unit (P/N: MPGL 06G H2)) using a peristaltic pump.
  • This system is capable of filtering about 7-10 liters before the pressure rises to 30-40 PSI and the 0.45 ⁇ m filter clogs.
  • EXAMPLE 6 CULTURE MEDIA CAPTURE COLUMN PROCEDURE
  • SP Sepharose Big Beads are strong ion exchangers with a large particle size (100-300 ⁇ m) which allows for high flow rates (1200-1800 cm/h) and quick abso ⁇ tion.
  • the ion exchange groups are coupled to the highly cross-linked agarose matrix through chemically stable ether bonds.
  • the strong ion exchange groups maintain full protein binding capacity over the whole operating pH range (4-12).
  • the primary objective of this procedure is to capture all the interferon in the media after culture and reduce the sample volume from about 10 liters to about 100 ml. This step is primarily for sample concentration and does little purification, although it removes the nutrients from the culture media and some small peptides.
  • the capture eluate is held for a maximum of about 20 minutes to one hour at room temperature before it is modified with Tween-20 and propylene glycol prior to being loaded onto the hydroxyapatite column.
  • This protease inhibitor is optional, but may be desired where the eluate is to be stored for any length of time.
  • Ceramic hydroxyapatite (HA) chromatography separates mixtures of biomolecules via adso ⁇ tion at a biocompatible calcium/phosphate [Cas(P04)6(OH)2] surface.
  • the structural arrangement of the calcium and phosphate ions provides for adso ⁇ tion of acidic, neutral and basic groups. Additionally, because the spatial arrangement of the functional groups is maintained in a rigid structure, HAC can accomplish separation of macromolecules by recognizing subtle structural differences between biomolecules. Because the support matrix also serves as the adsorbent, there are no chemically attached ligands to leach during the separation procedure. Ceramic HA can also be treated with 1 M NaOH, for effective cleaning and sterilization in one step.
  • HAC HSA
  • Typical yields are between about 80% ⁇ 8% by bioassay and between about 75% and 85% by an interferon ELISA assay (an average of at least 6 runs).
  • the procedure can be successfully scaled up to large volume preparations.
  • the specific conditions for carrying out HAC are as follows:
  • Equipment BioCad Chromatography Workstation or equivalent.
  • Resin Ceramic Hydroxyapatite, Type A, 40 ⁇ m, by American International Chemical, Inc. HAKS4 or its equivalent.
  • Load level a maximum of about 58 mg of total protein per ml of HA (as measured by the Pierce BCA assay of the culture medium (e.g., about 57 liters of original culture for 22 ⁇ m x 100 min 38 ml HAC column).
  • Flow rate Load and Wash at 720 cm3/hr. (Linear flow rate ).
  • Buffers are warmed to about 22° ⁇ 3°C, about 0.2 ⁇ m filter sterilized and sparged with helium or nitrogen (except for 1 M NaOH and 1 M MES pH 6.5) for a minimum of about 30 minutes prior to use.
  • a pool of crude interferon, such as the cation exchange capture is used for HAC.
  • the capture pool is already at about 10% PG and 0. 1% Tween-20, and is adjusted to a final pH of about 6.5 ⁇ 0.1 using about 2M TRIS-Base, about pH 8.8. If the pH is exceeded, about IM MES, about pH 6.5 is used to adjust the pH down to about 6.5. None shake or agitate the capture pool because of the potential to denature the product.
  • the crude interferon is applied to the HAC column.
  • An initial wash of about 100 mM MES, about pH 6.5, about 200 mM NaCl, about 10% PG, about 0.1-0.5% Tween-20 (10CV) is followed by a drop to about pH 5.0 with about 50 mM NH4OAc, about pH 5.0, about 10% PG, about 0.1-0.5% Tween-20 (linear gadient over 10CV).
  • the column is washed with about 50 mM NH4 ⁇ Ac, about pH 5.0, about 10% PG, about 0. 1-0.5% Tween-20 solution (5CV in forward direction, followed by 5CV in the reverse direction to reduce the volume of elution).
  • Elution of the partially purified interferon mixture is accomplished in reverse direction at about 360 cr /hr using a step elution of about 50 mM Na3PO4, about pH 5.0, about 200 mM Na-Cl, about 10% PG (v/v), about 0.1 -0.5% Tween-20 ( 10CV).
  • This process yields a concentrated protein pool of a partially purified interferon mixture, which is preferred for the following size exclusion chromatography step.
  • approximately 98% of the human serum albumin (HSA) contaminant is removed as well as other contaminating proteins and peptides.
  • this initial wash can also be performed with about 10 mM TRIS-HCI, about pH 7.4, about 10% PG, and about 0.1% Tween-20.
  • TRIS-HCI about pH 7.4, about 10% PG, and about 0.1% Tween-20.
  • the manufactures of the HAC packing material typically recommend against the use of pH levels lower than 5.5. Quite su ⁇ risingly, however, it has been discovered that the use of lower pH levels greatly assists in the purification of the partially purified interferon product and that this lower pH of about 5.0 does not negatively impact the HAC column material.
  • HAC eluate at least within about 2 days and more preferably within about 8 hours and most preferably within about 2 hours after elution. It should be understood that immediate use of the HAC eluate is recommended, i.e., within about two hours of elution, because of the possible appearance of a new peak in RP-HPLC after one week storage, although bioassay and ELISA indicate no change in the interferon when used immediately. When using immediately, HAC eluate is stored at about 4°C to about 8°C until loaded onto the SEC column to minimize proteolysis.
  • Superdex-75 prep grade is a highly resolving size exclusion media with average particle size of 34 ⁇ m. It is a composite of cross-linked agarose and dextran and is -useful for the separation of proteins with molecular weights between approximately 3 kDa and 70 kDa. In a typical SEC performed as described below, higher molecular weight contaminants including HSA elute prior the natural mixture of type I INF or interferon and lower molecular weight contaminants elute after the natural mixture of type I INF or interferon.
  • the first purification step, HAC removes about 98% of the human serum albumin which is present in the crude material. It is essential that most, if not all, of the human serum albumin be removed early in the purification process because of its ability to bind to many chromatographic resins and also to the natural mixture of type I INF or interferon. Early attempts to remove human serum albumin under non non-dissociating, non-denaturing conditions did not succeed. However, it has been quite su ⁇ risingly discovered that interferon subtype proteins associated with human serum albumin are separated by utilizing dissociating non-denaturing conditions.
  • dissociating, non-denaturing conditions e.g. about 10% propylene glycol, about 0.01% Tween-20
  • a partially purified natural mixture of type INF or interferon is successfully separated from the human serum albumin.
  • a reference standard is not necessarily required prior to running the SEC, and the HAC pool is loaded without any modification. Using this technique, only trace amounts of human serum albumin are found in the resulting interferon subtype eluate from the SEC.
  • the major contaminants after SEC are believed to be thrombospondin 1 -precursor fragment in the about 5-10 kDa region on SDS-PAGE gel.
  • Purity assessment of the SEC pool by SDS-PAGE and coomassie stain is not preferred because of the preferential dye binding properties of the thrombospondin- 1 precursor fragments.
  • Purity by RP-HPLC is about 50% to about 80% or greater.
  • Tween-20 from about 0.01 to about 0.05% can be used to provide enhanced separation of a calprotectin dimer contaminant present, so that slightly better resolution is obtained. There is no difference seen between about 0.01% and about 0.05%
  • Equipment A low pressure LC system (ISCO Protearn LC system 210 or equivalent) equipped with a UV detector operating at 280 nm and a chart recorder or preferably an appropriate data handling system.
  • Resin Superdex-75 by Pharmacia or its equivalent.
  • Bed volume 50 mm X 955 mm (1850ml). It should be understood that a column length of about 950 mm or greater is needed to obtain the appropriate resolution of interferon subtypes from contaminants.
  • Load level approximately 2 to 5% of Bed Volume and more preferably about 2.7% or less (i.e. 50.0 ml 1850 ml bed volume is optimal.
  • Flow rate about 3.0 ml/min.
  • SEC buffer about 50 mM Tris-HCI, about pH 7.4, about 10% propylene glycol, about 0.05% Tween-20 (Pierce Ultra Pure), about 50 mM NaCl, is sterile filtered at about 0.22 ⁇ m, stored at about 4°C prior to use and sparged (helium or nitrogen) for about 30 minutes prior to run. Column temperature: about 4-8°C. Detector. UV at 280 nm.
  • Fraction collection A fraction collector was set to start at about 5.25 hours after the start of isocratic elution. Fractions of 2.5 min or 7.5ml are collected and a total of 60 fractions are collected. Under these conditions, fractions 14-44 contain the natural mixture of type I INF or interferon.
  • the HAC fraction is loaded onto the column without any modification via line B of the gradient former at about 3.0 ml/min.
  • the separation is performed isocratically at about 3.0 ml/min and fractions 14-44 are collected.
  • the SEC eluate can be stored at about -80°C for up to about 1 week before continuing the purification process.
  • Anion exchange is suitable as a step for IFNs from contaminating proteins due to the acidic nature of the interferons (pl ⁇ 7.0).
  • Tris buffer at pH 7.5 natural IFNs carry a net negative charge (except for IFN- ⁇ ) and can be eluted and purified using a competing ion such as chloride.
  • the AX resin, Source-30Q, Pharmacia, or its equivalent uses quaternary amino groups, and is appropriate for this application because of its wide pH stability, temperature, and flow rate ranges (pH 2-12, 4-40°C 300-1000 crn/hr).
  • the partially purified interferon mixture obtained from SEC is adsorbed onto the 30Q matrix at low ionic strength and the interferon proteins are eluted in the reverse order of their acidity.
  • This can be accomplished for example by a linear gradient with a competing ion such as Cl or a 70 column volume wash with a buffer (about 50mM Tris-HCI, about pH 7.5, about 0.05% Tween-20) followed by a step elution with 50mM Tris, 0.01% Tween-20, 200 nM NaCl, pH 7.5.
  • the purity of the AX eluate averaged between about 95% to about 98% as measured by densitometric analysis of reducing and non-reducing SDS-PAGE and with RP-HPLC.
  • Equipment Alta ExplorerTM chromatography work station or its equivalent with Frac-900TM fraction collector or its equivalent. Resin, Source-30QTM from Pharmacia Biotech, product number 17-1275, or equivalent.
  • Bed Volume 7.5 mm X 50 mM Altech PEEK column with 2.21 ml bed volume.
  • Flow rate Load at about 722 cm/hr. Wash and elute at 361 cm/hr. Load: 1 mg protein/ml of bed volume. A lower load may slightly improve the recovery of interferon subtypes, but will increase the column size and subsequent elution volume. Injection volume is variable.
  • Buffers Equilibration and elution buffers are filtered through a C-18 RPLC column and vacuum hit through sterile 0.22 ⁇ m cellulose acetate filter prior to adding
  • the SEC pool of IFN is diluted 1 :1 with deionized water, bringing the salt concentration to about 25 mM, the pH is checked to ensure that it is at about 7.4 and the SEC pool is applied to the 30Q column.
  • Some loss of interferon occurs in the flow through (11-23% by ELISA and bioassay). The nature of this loss is not fully understood, but it is probably due to loss the IFN- ⁇ , IFN- ⁇ l4 and IFN- ⁇ 21 which may not bind to the Source-30Q resin because of their isoelectric points.
  • This column is washed with about 10-70 CV of about 50 mM Tris-HCI, about pH 7.5, about 0.05% Tween-20.
  • This wash removes the main contaminants which are thrombospondin- 1 -precursor protein fragments and IL-6 and other low molecular weight peptides Elution. is carried out via a linear gradient of 0 to about 200 mM NaCl, about 50 mM
  • Tris-HCI about pH 7.5, about 0.05%) Tween-20 (60CV) or more preferably, a step elution using about 200 mM NaCl, about 50 M Tris-HCI, about pH 7.5, about 0.01% Tween 20.
  • fractions 4-40 are pooled to provide maximum interferon subtype recovery, but fractions 16-38 are pooled to provide maximum purity.
  • HIC hydrophobic interaction chromatography
  • reversed phase chromatography is a technique for separating biomolecules by their degree of hydrophobicity. Since the elution of proteins in HIC uses a decreasing salt gradient, the biological activity of the eluted proteins is usually preserved.
  • HIC resins There are many different types of HIC resins; however, the natural mixture of type I INF or interferon is found to have the best separation from contaminants using a weakly hydrophobic propyl ligand with a silica matrix, such as Bakerbond Wide Pore Hl-propyl (40 ⁇ m), JT Baker L05082 or L05083 or equivalents.
  • This resin offers a high binding capacity along with the necessary rigidity to withstand high flow rates for quick and consistent processing. Due to the small amount of resin required for each batch, the used resin can be discarded after each run, eliminating the need for cleaning and sterilization thereby reducing overall procedural costs and time.
  • the HIC method described herein uses a high concentration of a non-chaotropic salt to bind the IFN subtypes to the HIC column. It is believed that the non-chaotropic salt has a stabilizing influence on the interferon subtype structures. Because the resin is silica based, it is coated by JT Baker with polyimines for bioprocessing use to prevent non-specific irreversible abso ⁇ tion of proteins to the resin. Phosphate buffer and sodium chloride are used to elute the natural mixture of type I INF or interferon. The use of this combination of phosphate buffer and sodium chloride eliminates the need for an additional buffer exchange step after the elution.
  • Flow Rate all at about 8.04 ml/min (240 cm/hr).
  • Buffer All buffers are sparged with helium or nitrogen for about 30 minutes prior to use.
  • Column temperature ambient 20-25°C.
  • Detector UV at about 280 nm. Fraction collection during the elution.
  • the IFN-containing SEC fractions are applied to the Hl-Propyl resin with in- line mixing (1 : 1.6) with about 2 M (NH 4 ) 2 SO4 (final salt concentration at about 1.25 M) followed by a 1.25 M (NH4)2SO425, mM Tris, pH 7.4 wash (10CV) allows full binding of the all IFN subtypes. If inline mixing is not possible, the salt can be slowly added directly to the SEC pool to a concentration of about 1.25M (NH4)2SO4. The composition of the mobile phase is then changed from about 25 mM TRIS, about pH 7.4, about 1.25 M (NH4) 2 SO4 to about 25mM Na3PO4, about pH 7.0, about 1.75 M NaCl through a linear gradient (10 CV). The column is washed with about 25 mM Na3PO4, about pH 7.0, about 1.75 M NaCl (60 CV) in order to wash off any residual (NH4)2SO4.
  • the natural mixture of type I INF or interferon is eluted from the column with a 5 column volume linear gradient from about 25 mM Na3PO4, about pH 7.0, about 1.75 M NaCl to about 25 mM Na3PO4, about pH 7.0.
  • the pH of the mobile phase is lowered to a final pH of approximately 2.2 using about 42.5 CV of about 0.01M HC1.
  • the salt drop and pH drop fractions are combined to form a final pool which has a buffer composition of approximately: about 450 mM NaCl, about 2-3 mM Na 3 PO4, about pH 6.4-6.6.
  • This method is reproducible and scalable with full subtype retention and high mass recovery (about 65% by anti-viral ELISA, about 67% by bioassay and about 75-80% by RP-HPLC).
  • the bioactivity is also retained with a purity level of greater than 95% by both RP-HPLC and reducing and non-reducing SDS-PAGE using coomassie equilibrium staining.
  • An independent laboratory has confirmed that the ammonium sulphate present in the final product was at background level and experiments have shown that the HIC eluate is stable to repeated freeze thaw cycle.
  • the natural mixture of interferon was characterized in several ways at each step in the procedure. Characterizations included protease assay (Universal Protease Assay, Boehringer Mannhein), BCA assay for total protein (Pierce, RD0125), ELISA for interferon omega ( ⁇ ) (Bender Wein, Austria), ELISA for IFN- ⁇ (Endogen, Pestka Biomedical Laboratories, BioSource International or Bender MedSystems), ELISA for IL-6 (CYT Immune, Inc., MD), reducing and non-reducing SDS-PAGE with coomassie (equilibration staining), silver staining or Western blot followed by densitometric analysis, antiviral bioassay, RP-HPLC, and amino terminal and tryptic fragments sequencing (with BioBrene/TFA filter) and mass spectrometry of individual RP-HPLC peaks. Summaries of the data obtained is provided as follows:
  • SAMPLE PREPAR ATION Begin with 5.0 X 10 6 IU/ml 1.0 mg/ml HSA NATURAL MIXTURES: OF TYPE I IFN OR INTERFERONTM formulated product. Aliquot 1 ml of NATURAL MIXTURES OF TYPE IFN OR INTERFERONTM into a wide-mouth amber autosampler vial. Add 667 ⁇ l of Mobile Phase B. Cap vial with appropriate septa (acetonitrile resistant, e.g. PTFE linear) and invert several times to ensure adequate mixing. Place samples in a chilled (4°C) autosampler prior to run. (Note: The final volume of prepared sample (1667 ⁇ l) should be adequate for a 999 ⁇ l sample injection.
  • the RP-HPLC method for HAS formulated of natural mixture of Type I IFN or interferon provides a tool for the determination of any primary structural changes which may occur over time.
  • the method is reproducible and yields excellent resolution of all subtypes present in the final interferon mixture.
  • This method can also be used to analyze products from different vendors currently being formulated with HSA.
  • the method performed well when analyzing Interferon Sciences material
  • AlferonTM This method confirms the claimed Alferon subtypes such as IFN- ⁇ 2, IFN— ⁇ 8.
  • RP-HPLC can be used as a "physical measurement" (e.g. the presence or absence of a compound), but not to determine biological activity of a compound.
  • a change in the RP-HPLC chromatogram is indicative of a conformational or structural change in the protein undergoing analysis.
  • Primary structural changes as well as conformational changes in one or more of natural mixture of Type I IFN or interferon subtypes may or may not result in a change in bioactivity.
  • RP-HPLC should be used in conjunction with mass spectroscopy for final determination of any structural changes that may occur over time.
  • mass spectroscopy for final determination of any structural changes that may occur over time.
  • RP-HPLC can be modified to allow the inco ⁇ oration of internal and external standards in order to monitor column performance.
  • phenylthiohydantoin norleucine PTH-Nle, SIGMA PI 877
  • 25 mg is mixed with 10 ml of Mobile phase B and stored at -
  • Fmoc-Nle SIGMA F 2917
  • 10 mg of Fmoc-Nle is weighted into a flask with 5 ml of 40% Mobile phase A/60%-Mobile phase B and warmed until solubilized. Then the volume is brought to 10 ml with

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Abstract

Cette invention porte sur des procédés visant à isoler des leucocytes des mélanges extrêmement purifiés d'interférons naturels de type I. Cette invention porte également sur des mélanges extrêmement purifiés d'interférons naturels de type I qui ressemblent à l'interféron naturel comprenant 9 sous-types tels que alpha-1, alpha-2, alpha-5, alpha-7, alpha-8, alpha-10, alpha-14, alpha-21 et oméga, donnant éventuellement naissance à 20 espèces moléculaires telles que alpha-1a, alpha-1new, alpha-2a, alpha-2b, alpha-2c, alpha-5, alpha-5LG, alpha-7, alpha-8a, alpha-8c, alpha-10a, alpha-14a, alpha14-b, alpha 14-c, alpha-14LG, alpha-21a, alpha-21b, alpha-21c, oméga and oméga LG.
EP99967753A 1998-12-31 1999-12-29 Procedes et composition de melanges naturels extremement purifies d'interferons de type i derives des leucocytes Withdrawn EP1140144A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US224895 1998-12-31
US09/224,895 US6350589B1 (en) 1998-12-31 1998-12-31 Compositions of highly-purified natural mixtures of type I interferon derived from leukocytes and methods
US09/229,039 US6433144B1 (en) 1999-01-12 1999-01-12 Compositions of highly-purified natural mixtures of type I Interferon derived from leukocytes and methods
US229039 1999-01-12
PCT/US1999/031198 WO2000039280A2 (fr) 1998-12-31 1999-12-29 Procedes et composition de melanges naturels extremement purifies d'interferons de type i derives des leucocytes

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