WO1999064441A1 - Procede de preparation de compositions pharmaceutiques exemptes de virus - Google Patents

Procede de preparation de compositions pharmaceutiques exemptes de virus Download PDF

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WO1999064441A1
WO1999064441A1 PCT/FI1999/000505 FI9900505W WO9964441A1 WO 1999064441 A1 WO1999064441 A1 WO 1999064441A1 FI 9900505 W FI9900505 W FI 9900505W WO 9964441 A1 WO9964441 A1 WO 9964441A1
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virus
interferon
ifn
filtration
solution
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PCT/FI1999/000505
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English (en)
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Hannele TÖLÖ
Jaakko Parkkinen
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Suomen Punainen Risti Veripalvelu
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Priority to EP99931279A priority Critical patent/EP1086120A1/fr
Priority to AU47834/99A priority patent/AU4783499A/en
Publication of WO1999064441A1 publication Critical patent/WO1999064441A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
    • A61L2/0017—Filtration
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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

Definitions

  • the present invention relates to the preparation of virus-safe pharmaceutical compositions of biologically active proteins.
  • the present invention concerns a method for preparing a virus-safe, liquid formulation of ⁇ -interferon, preferably multicomponent ⁇ - interferon, having extended shelf-life.
  • the present invention also relates to the use of non- ionic detergents as stabilizers of pharmaceutical compositions and to virus-safe multi- component ⁇ -interferon solutions which can be used as injectables in the treatment of diseases.
  • compositions of biologically active proteins must be virus-safe, i.e. they must be free from any contaminating, potentially pathogenic viruses and other infectious agents. Further, such pharmaceutical compositions should have extended shelf-life providing for their use over a prolonged period of time. In the following, the questions of virus-safety and shelf-life of proteineous pharmaceutical compositions will be discussed with particular reference to interferon formulations. However, the principles are generally applicable to physiologically active substance originating from human or animal blood, urine or internal organs and to corresponding recombinant proteins produced in cultured animal cells or transgenic animals.
  • IFN- ⁇ Human alpha-interferons
  • Human leukocytes and lymphoblastoid cells are known to produce several IFN- ⁇ subtypes in culture when induced by Sendai virus (Cantell et al., Methods Enzymol. 78, 29-38, 1981, Mizrani, Methods Enzymol. 78, 54-68, 1981).
  • Purified multicomponent IFN- ⁇ drugs are used in the treatment of various diseases, including neoplastic and viral diseases.
  • multicomponent IFN- ⁇ drugs have therapeutic benefits in comparison with recombinant IFN- ⁇ drugs produced in bacteria, which only contain a single IFN- ⁇ subtype.
  • Commercial production of human multicomponent IFN- ⁇ comprises culturing human leukocytes or lymphoblastoid cells and inducing them with Sendai virus. These products therefore carry a risk of virus contamination.
  • Blood-borne viruses potentially present in leukocytes and serum or its fractions used in the culture medium include Hl-viruses, hepatitis C and B viruses and small non-enveloped viruses, such as parvovirus B19, which is resistant to many physicochemical treatments.
  • Lymphoblastoid cell lines may harbour e.g. retroviruses. Production of IFN- ⁇ and other biologically active proteins in animal cell cultures or in transgenic animals also carries a risk of viral contamination.
  • An effective method for the removal of viruses of diverse physicochemical properties is filtration with membranes with high virus retentive capacities, also known as nanofiltration or virus filtration.
  • the particular advantage of filtration is that it will also remove viruses, such as non-enveloped viruses, and other infectious agents, such as those causing transmissible spongiform encephalopathies ("prions"), which exhibit resistance to conventional treatments based on the use of heat and chemicals (physicochemically resistant agents).
  • stabilizers are typically added to solutions containing the purified biologically active protein.
  • stabilizers will also prevent aggregation of the proteins and, thus, provide extended shelf-life.
  • Albumin is the most common stabilizer used, e.g., in multicomponent IFN- ⁇ products and it is employed in many of the commercial preparations (Alfanative®, Alferon® N, Wellferon®).
  • albumin as a stabilizer in IFN- ⁇ products may cause at least two problems.
  • albumin has been reported to result in the formation of albumin-IFN aggregates in the product, which may be antigenic and result in the formation of antibodies against IFN- ⁇ (Braun et al., Pharm. Res. 14, 1472-1478, 1997). These problems have been identified with bacterial recombinant IFN- ⁇ products.
  • the use of albumin as a stabilizer decreases the ability of the filter to remove viruses, since it has been shown that virus removability of a virus removal filter decreases with increasing concentration of coexisting protein
  • the coating of the filter with albumin will not reduce adsorption of the proteins to other surfaces being in contact with the product, such as tubing, collecting vessels, vials and stoppers.
  • It is another object of the invention is to provide a new use of non-ionic detergents as stabilizers for liquid formulations of biologically active proteins, such as IFN- ⁇ , which can be filtered with a virus removal filter with improved yield and capacity and used as injectables.
  • the present invention is based on the finding that by using a non-ionic detergent as a stabilizer of pharmaceutical compositions comprising biologically active proteins and by adding said stabilizer to the formulation before virus filtration, the yield and capacity of virus filtration can be greatly increased.
  • This finding was surprising since it is known that non-ionic surfactants, like polysorbate 80, have very low critical micelle concentrations (CMC).
  • CMC critical micelle concentrations
  • non- ionic surfactants form micelles with varying sizes, which penetrate very slowly e.g. dialysis membranes.
  • non-ionic detergents are added to pharmaceutical compositions in concentrations above the CMC before virus removal filtration to provide stabilized proteineous formulations, for example multicomponent IFN- ⁇ formulations, which are essentially free from substances (including viruses and prions) having a size in excess of 10 to 40 nm, in particular 10 to 20 nm, and normally being retained on a virus filter.
  • the present method for preparing virus-safe pharmaceutical compositions of biologically active proteins is characterized by what is stated in the characterizing part of claim 1.
  • the method for stabilizing pharmaceutical compositions of purified leukocyte ⁇ - interferon is characterized by what is stated in the characterizing part of claim 13 and the virus-safe ⁇ -interferon solution is characterized by what is stated in the characterizing part of claim 15.
  • a multicomponent IFN- ⁇ solution stabilized according to the present invention with a non-ionic detergent exhibits improved stability.
  • multicomponent IFN- ⁇ formulations stabilized with a non-ionic detergent do not contain albumin-IFN complexes, which are formed in albumin-containing formulations and are suggested to be harmful in recombinant IFN- ⁇ products.
  • albumin By replacing albumin with a non-ionic detergent as a stabilizer, an IFN- ⁇ solution can be filtered with a virus removal filter without plugging of the filter.
  • a non- ionic detergent for albumin, it is possible to filter IFN- ⁇ solution with improved yield and capacity with a virus removal filter.
  • the present invention not only increases the yield of filtration, it also prevents losses caused by adsorption of protein from the filtrate to other surfaces being in contact with the product, such as tubing, collecting vessels, vials and stoppers.
  • the non-ionic detergent in the composition before filtering, no pretreatment of the filter is necessary. In fact, test have shown, that such a pretreatment will not improve the yield to any discernible extent.
  • Figure 1 shows the adsorption of IFN- ⁇ to glass in the presence of different stabilizers
  • Figure 2 shows the occurrence of albumin-IFN complexes in solutions stabilized with albumin and the lack of aggregates in solutions stabilized with polysorbate 80
  • Figure 3 depicts the virus filtration flow rates of purified IFN- ⁇ solutions stabilized by polysorbate 80 and albumin, respectively.
  • a non-ionic detergent is added to a solution of purified biologically active protein, which is subsequently filtered with a virus removal filter having a pore size of about 10 to 40 nm and then optionally sterile filtered to obtain a virus-safe, sterile and stable protein solution.
  • biologically active proteins covered by the present invention extends to all therapeutically used proteins which may harbour viruses and which are filtered with a virus removal filter.
  • Such proteins generally have a molecular weight of less than 180,000 D and include coagulation factors and their activated forms (e.g. factor IX, factor VII), proteinases, their activated forms and proteinase inhibitors (e.g. protein C), growth factors and colony stimulating factors (e.g. IGF-1, G-CSF, GM-CSF), neurotrophic factors (e.g. NGF, GDNF, NT-3), hormones (e.g. erythropoietin, growth hormone) and other proteins modifying the biological response of cells (e.g. interferons and interleukins).
  • coagulation factors and their activated forms e.g. factor IX, factor VII
  • proteinases e.g. protein C
  • growth factors and colony stimulating factors e.g. IGF-1, G-CSF, GM-CSF
  • non-ionic detergents in various pharmaceutical compositions is known er se. It has also been suggested in the art to use polysorbate 80 instead of albumin as a stabilizer of a recombinant IFN- ⁇ 2a product in order to prevent formation of albumin-IFN aggregates (Hochuli, J. Interferon Cytocine Res. 17, Suppl. 1, S15-S21, 1997). Liquid ⁇ - and ⁇ - interferon compositions containing non-ionic detergents are also disclosed in EP Patent Application No. 0 736 303 A2 and WO 89/04177. However, all the citations are completely silent about the incorporation of a non-ionic detergent into a pharmaceutical composition prior to virus-filtration.
  • non-ionic detergents are used as stabilizers of multicomponent IFN- ⁇ formulations subjected to virus filtration for removing any agents retained on filters having a pore size of 10-40 nm.
  • These compositions comprise purified leukocyte and lymphoblastoid interferons containing two or more of the following IFN- ⁇ subtypes: ⁇ l, ⁇ 2, ⁇ 4, ⁇ 7, ⁇ 8, ⁇ lO, ⁇ l4, ⁇ l7 and ⁇ 21.
  • Human leukocyte interferon has been shown to contain at least nine IFN- ⁇ subtypes (Nyman et al., Biochem. J.
  • lymphoblastoid interferon contains the same or similar subtypes (Zoon et al., J. Biol. Chem. 267, 15210-15216, 1992). Part of the subtypes secreted by the producer cells may be lost during purification, depending on the purification process employed (US Patent 5,503,828).
  • Multicomponent IFN- ⁇ can be produced in leukocyte or lymphoblastoid cell cultures by Sendai virus induction.
  • IFN- ⁇ subtypes with close structural similarity to the natural subtypes can be produced by recombinant DNA technology in cultured animal cells or in transgenic animals.
  • the process for manufacturing a highly purified drug substance may consist of precipitations, filtrations and chromatographic steps. Purification methods of multicomponent IFN- ⁇ employing monoclonal or polyclonal antibodies have also been disclosed.
  • the manufacturing process may contain additional virus inactivation steps, such as treatment with low pH and solvent/detergent treatment.
  • IFN- ⁇ composition and methods for its production from human peripheral blood leukocytes are disclosed in, e.g. US Patents Nos. 5,503,828 and 5,391,713, the contents of which are herewith incorporated by reference.
  • a purification process yielding all major IFN- ⁇ subtypes is described in Example 2. Generally, it comprises, e.g., the step of contacting a solvent/detergent treated composition with at least two monoclonal mouse IgG antibodies having complementary subtype specificities in an immunoadsorption step. The ⁇ -interferon subtypes bound by the monoclonal antibodies are eluted and the eluate is purified and filtered on a virus removal filter.
  • compositions which can be subjected to virus removal filtration can be produced by methods known er se, for example by isolating from human or animal blood or by recombinant DNA technology in cultured cells or transgenic animals.
  • a formulated protein solution is prepared by diluting a calculated amount of the purified biologically active protein with a formulation buffer containing polysorbate 80 or another non-ionic detergent in an amount, which gives a final concentration of 0.05 to 1 g/1, preferably about 0.1-0.5 g/1, of the non-ionic detergent.
  • the degree of purity of the protein is advantageously at least about 90 % .
  • the formulated solution may be prefiltered with a 0.04-0.2 ⁇ m filter and thereafter filtered with a virus removal filter having a preferred pore size of 10-40 nm.
  • the non-ionic detergent does not cause any plugging of the filter and, depending on the molecular size of the protein, the filtration can be carried out with a constant pressure without any decrease in the filtrate flux and thus with high capacity and constant removability of viruses.
  • Two virus filters may be used sequentially, which improves virus removal.
  • the recovered filtrate is filtered with a sterile filter and filled in vials, syringes or other containers compatible with parenteral injectables. It is also possible to carry out the virus filtration and sterile filtration in reversed order.
  • a virus removal filter includes filters suitable for the removal of viruses from pharmaceutical proteins solutions.
  • the size of the pores or perforations in the filter should be small enough to effectively remove even small non- enveloped viruses, such as parvoviruses.
  • the proper pore size can be assessed by spiking experiments with model viruses, in which at least 4 log, preferably at least 6 log, of model viruses with a size of ca 20 to 40 nm should be removed. Based on such tests, the theoretical pore sizes of the virus removal filters can be estimated to be about 10 to 40 nm, preferably about 10 to 20 nm.
  • virus filters capable of reducing the concentration of model viruses at the above mentioned spiking tests with at least 4 log, are considered to have a "high virus retentive capacity". It is particularly important that the filters used have such capacity also in relation to small non-enveloped viruses.
  • the buffer of the liquid formulation is less critical and may be an inorganic buffer or organic buffer.
  • the pH of the buffer may be in the range of 4.5-7.5, and the buffer may contain other substances, e.g. inorganic salts, sugars, amino acids, polyols or cyclodextrins.
  • Other stabilizers can be added to IFN- ⁇ solution after the virus filtration step.
  • the activity of IFN- ⁇ solution to be filtered with a virus removal filter may be close to that in the final product or it may be considerably higher. In the latter case, the solution is diluted after virus filtration.
  • the activity of IFN- ⁇ in the final product is selected based on several variables, including the disease to be treated, therapeutic regimen and administration system. Generally, the activity of IFN- ⁇ solution before virus filtration is in the range of 3 to 50 mill. IU/ml.
  • non-ionic detergents to be used as a stabilizer include polyoxyethylene-based detergents, such as polyoxyethylene sorbitan monooleate (polysorbate 80), polyoxyethylene sorbitan monolaurate (polysorbate 20), polyoxyethylene lauryl ethyl (laureth 4) and polyoxyethylene, polyoxypropylene block polymer (poloxamer 188).
  • polyoxyethylene-based detergents such as polyoxyethylene sorbitan monooleate (polysorbate 80), polyoxyethylene sorbitan monolaurate (polysorbate 20), polyoxyethylene lauryl ethyl (laureth 4) and polyoxyethylene, polyoxypropylene block polymer (poloxamer 188).
  • Polysorbate such as polysorbate 80 is most preferred.
  • Polysorbate 80 as well as the other non-ionic detergents are used at concentrations in excess of the critical micellar concentration, in the case of polysorbate 80 typically about 0.05 to 1 g/1.
  • a preferred range is 0.1-0.5 g/1, and most preferred concentration about 0.2 g/1.
  • the non-ionic detergent used has a low peroxide number, so as to prevent any harmful oxidation reactions in the pharmaceutical formulations.
  • the peroxide number is less than 5.0 mEq/kg tested according to Ph. Eur. 1997.
  • an antioxidant can be added to the formulation in order to prevent oxidation of IFN- ⁇ .
  • the IFN- ⁇ concentration was measured by a time-resolved fluoroimmunoassay (FIA) on microtitre plates.
  • FSA fluoroimmunoassay
  • the IgG fraction of a bovine antiserum against human leukocyte IFN- ⁇ was used in capturing and a mixture of two Eu-labelled mouse IgG monoclonal antibodies to IFN- ⁇ for detection.
  • the monoclonal antibodies were the same as used in the purification of IFN- ⁇ (Example 1). The details of the assay have been described elsewhere (Ronnblom et al., APMIS 105, 531-536, 1997).
  • IFN- ⁇ concentration was expressed as IU/ml using a laboratory standard, which was calibrated by the virus plaque reduction assay against the International Reference Preparation of Interferon, Human Leukocyte 69/19 (NIBCS, U.K.).
  • the antiviral activity of the IFN was determined by a virus plaque reduction assay in 35 mm petri dishes using Human Epithelial 2 (HEp2) cells challenged with Vesicular stomatitis virus (VSV).
  • HEp2 Human Epithelial 2
  • VSV Vesicular stomatitis virus
  • EMEM fetal calf serum
  • FCS fetal calf serum
  • aureomycin 0.004% The samples were assayed as triplicates at four dilutions in at least two assay series.
  • Virus control dishes without IFN were included in each assay series. After incubation of overnight at 37 °C in 3-4% CO 2 atmosphere the solutions were removed from the confluent cell layers and 150-200 PFU of VSV in 1 ml of EMEM was added. After incubation of 40-45 min the virus was removed and cells were overlayed with 2 ml of agar 0.8% in EMEM. After overnight incubation the virus plaques were calculated.
  • One unit of IFN activity is the highest dilution of the sample, which inhibits 50% of the virus plaques as compared to the virus control. Interferon activity was expressed in International
  • Units using a laboratory standard, which was calibrated against the International Reference Preparation of Interferon, Human Leukocyte 69/19 (NIBCS, UK).
  • Total protein Total protein concentration was measured according to Lowry using human albumin as a standard (Total Protein Standard, Finnish Red Cross Blood Transfusion Service, Helsinki, Finland).
  • the membrane was washed and incubated with peroxidase-conjugated rabbit anti-bovine IgG (Jackson Immunoresearch Laboratories, PA, USA). After washing, the positive bands were visualized by using 4-chloro-l-naphthol as the peroxidase substrate.
  • Polysorbate 80 concentration was measured by a colorimetric method (Milwidsky, Analyst 94, 377-386, 1969).
  • This example describes the production of a high purity leukocyte IFN- ⁇ drug substance which was used in the stabilization and filtration examples (Examples 2-5).
  • the production of crude interferon was carried out in leukocyte cultures induced by Sendai virus essentially as described before (Cantell et al., Methods Enzymol. 78, 29-38, 1981). Residual cells in the culture supernatant were removed by microfiltration and the filtrate was concentrated 20-fold by ultrafiltration.
  • the crude IFN concentrate was filtered through 1.2 ⁇ m and 0.22 ⁇ m filters and treated with 0.3% tri(n-butyl)phosphate and 1% polysorbate 80 for 16 h at 26 °C (solvent/detergent treatment). The solution was applied to an immunoadsorbent column containing two monoclonal antibodies against IFN- ⁇ coupled to CNBr-Sepharose 4FF gel.
  • the monoclonal antibodies have complementary binding specificities and together bind all major IFN- ⁇ subtypes.
  • the immunoadsorbent column was washed extensively and the bound IFN- ⁇ was eluted with buffer adjusted to pH 2. The eluate was neutralized and concentrated about 30-fold by ultrafiltration. The concentrated eluate was applied to a Superdex 75 gel filtration column equlibrated and eluted with PBS. The IFN- ⁇ containing fractions were pooled and the purified drug substance thus obtained was stored frozen at -70 °C.
  • the purified drug substance was analyzed for IFN- ⁇ subtype composition by using procedures described in detail elsewhere (Nyman et al., Biochem. J. 329, 295-302, 1998). It was found to contain the subtypes ⁇ l, ⁇ 2, ⁇ 4, ⁇ 7, ⁇ 8, ⁇ lO, ⁇ l4, ⁇ l7 and ⁇ 21.
  • Example 2
  • Short-term stabilizing effect of various stabilizers was determined by assessing the adsorption of IFN- ⁇ onto glass.
  • Purified leukocyte IFN- ⁇ bulk drug was diluted in polypropylene vials to a final concentration of 3 mill. IU/ml (0.02 g/1) in PBS containing one of the following stablizers:
  • polyoxyethylene lauryl ether (laureth 4, Brij® 35, CAS-9002-92-0)
  • polyoxyethylene sorbitan monooleate polysorbate 80, Tween® 80, CAS-9005-65-6)
  • polyoxyethylene, polyoxypropylene block polymer (poloxamer 188, Pluronic® F-68, CAS-9003-11-6) 4. human serum albumin
  • Laureth 4, polysorbate 80 and poloxamer 188 were used at final concentrations of 0.1, 0.2, 0.5 and 1.0 g/1. Albumin was added to a final concentration of 0.5, 1.0, 1.5, and 2.0 g/1.
  • the IFN- ⁇ bulk drug was diluted in PBS. Samples were taken from the formulated solutions immediately after mixing for the determination of IFN- ⁇ concentration, and 100 ⁇ l of the formulated solutions were transferred into glass vials. The vials were kept for 20 h at at room temperature (23 °C). Samples were taken for IFN- ⁇ concentration determination. The results are shown in Figure 1. Adsorption was determined as the difference between the initial and final concentration of IFN- ⁇ in the vials.
  • IFN- ⁇ About 30 % of IFN- ⁇ was adsorbed onto the glass vials in the absence of any stabilizer (Fig. 1).
  • the stabilizers studied prevented the adsorption of IFN- ⁇ to a different extent.
  • Polysorbate 80 was most effective followed by laureth 4, albumin and poloxamer 188.
  • IFN-containing aggregates were studied by Western blot analysis under non- reducing conditions. Highly purified leukocyte IFN- ⁇ was incubated in glass vials in PBS containing polysorbate 80 or albumin for 20 h at at 23 °C.
  • Figure 2 shows the Western blot of the samples containing 0.1 g/1 (lane 3), 0.2 g/1 (lane 4), and 0.5 g/1 (lane 5) of polysorbate 80, and 0.5 g/1 (lane 6), 1.0 g/1 (lane 7) and 1.5 g/1 (lane 8) of albumin. Lanes 1 and 2 show negative and positive IFN- ⁇ aggregate controls, respectively. In polysorbate 80-containing solutions only bands corresponding to IFN- ⁇ monomers and dimer were seen.
  • the intensity of the dimer band was weaker at polysorbate 80 concentrations 0.2 g/1 and 0.5 g/1 than at 0.1 g/1.
  • dimer bands were more intensive and additionally, bands with higher molecular weight corresponding to albumin-IFN complexes were seen.
  • polysorbate 80 formulations no bands corresponding to higher molecular weight complexes could be detected.
  • Purified leukocyte IFN- ⁇ was diluted to the activity of 5 mill. IU/ml (40 ⁇ g/ml) in PBS containing either 0.2 g/1 polysorbate 80 or 1 g/1 albumin.
  • the formulated solutions were prefiltered with a 0.1 ⁇ m filter and subjected to virus filtration by using Planova 15N filters (Asahi Chemical Industry Co, Japan). Filtrations were carried out in tangential flow mode at room temperature with a constant pressure of 0.8 bar. The system was pressurized with nitrogen gas. At the end of the filtration the virus filter was washed with formulation solution in the dead-end mode in order to recover all product from the filter system. Pressure, temperature and the mass of the filtrate were recorded during filtration. Samples were taken from the formulated solutions, after prefiltration, after virus filtration, and after sterile filtration for the determination of IFN- ⁇ concentration, polysorbate 80 and total protein and Western blot assay.
  • Table 1 indicates the yield of IFN- ⁇ in the manufacture of a virus-filtered finished product by using polysorbate 80 (0.2 g/1) or albumin (1 g/1) as a stabilizer.
  • the yield of IFN- ⁇ in the virus-filtered and sterile-filtered solution was consistently better in the presence of 0.2 g/1 polysorbate than in the presence of 1 g/1 albumin.
  • Most of the IFN- ⁇ loss in albumin solutions took place during virus filtration, whereas there was no significant loss of IFN- ⁇ in polysorbate solution at the corresponding step.
  • the recovery of polysorbate 80 was 99 % in filtrate of the virus filtration, indicating that there was no retention tendency of polysorbate during virus filtration.
  • the recovery of albumin in the filtrate was 87% indicating that albumin was retented by the filter.
  • Figure 3 depicts Planova 15N filtration flow rates of purified IFN- ⁇ solutions stabilized by polysorbate 80 or albumin.
  • Purified leukocyte IFN- ⁇ 40 ⁇ g/ml
  • PBS containing 0.2 g/1 polysorbate 80 (open circles) or 1.0 g/1 albumin was filtered with Planova 15N filter at a constant pressure of 0.8 bar in tangential flow mode.
  • the filtration rate remained constant in the presence of polysorbate 80 at least during filtration of 200 1/m 2 , whereas it was reduced by about 80% in the presence of 1 g/1 albumin already after filtration of 20 1/m 2 .
  • a formulated IFN- ⁇ bulk solution was prepared by adding to a suitable container PBS and polysorbate 80, mixing them, and adding purified multicomponent IFN- ⁇ so that the desired IFN- ⁇ activity was obtained in the calculated final volume of PBS containing 0.2 g/1 of polysorbate 80.
  • the formulated IFN- ⁇ solution was mixed carefully and prefiltered with a 0.1 ⁇ m filter.
  • the prefiltered IFN- ⁇ solution was filtered through a virus filter (Planova 15N, Asahi) at a constant pressure of 0.9 bar in a dead-end mode.
  • the filtrate was recovered and filtered with a 0.1 or 0.22 ⁇ m sterile filter and filled aseptically into the final containers.
  • the stability of the virus-filtered IFN- ⁇ finished product manufactured according to Example 4 was studied at 6 °C and at 25°C up to six months. The results are given in Table 2.

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  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Dermatology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un procédé de préparation de compositions pharmaceutiques d'une protéine biologiquement active, en particulier des compositions d'interféron à constituants multiples. Le procédé consiste à ajouter à une solution de la protéine un détergent non ionique en quantité suffisante pour conférer à la composition pharmaceutique une durée de conservation prolongée. Le procédé consiste ensuite à soumettre la solution contenant le détergent non ionique à une filtration réalisée avec un filtre d'excrétion de virus ayant une dimension des pores comprise entre 10 et 40 nm, puis à récupérer le filtrat. On obtient ainsi, par exemple, une composition d'interféron alpha à constituants multiples exempte de virus. Cette composition contient, comme stabilisateur, une quantité du détergent non ionique supérieure à la concentration de tensioactifs critique du détergent, et est sensiblement exempte des substances retenues dans un filtre d'excrétion de virus ayant une grande capacité de rétention de virus.
PCT/FI1999/000505 1998-06-10 1999-06-09 Procede de preparation de compositions pharmaceutiques exemptes de virus WO1999064441A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP99931279A EP1086120A1 (fr) 1998-06-10 1999-06-09 Procede de preparation de compositions pharmaceutiques exemptes de virus
AU47834/99A AU4783499A (en) 1998-06-10 1999-06-09 Method for preparing virus-safe pharmaceutical compositions

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI981337A FI106465B (fi) 1998-06-10 1998-06-10 Menetelmä virusturvallisten farmaseuttisten koostumusten valmistamiseksi
FI981337 1998-06-10

Publications (1)

Publication Number Publication Date
WO1999064441A1 true WO1999064441A1 (fr) 1999-12-16

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FI1999/000505 WO1999064441A1 (fr) 1998-06-10 1999-06-09 Procede de preparation de compositions pharmaceutiques exemptes de virus

Country Status (4)

Country Link
EP (1) EP1086120A1 (fr)
AU (1) AU4783499A (fr)
FI (1) FI106465B (fr)
WO (1) WO1999064441A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003274941A (ja) * 2002-03-15 2003-09-30 Aventis Behring Gmbh ナノろ過によってタンパク溶液からウイルスを分離する方法
WO2005039652A2 (fr) * 2003-10-09 2005-05-06 Prodotti Chimici E Alimentari Spa Procede permettant d'eliminer les agents infectieux de l'encephalopathie spongiforme transmissible (tse) de la bile et de ses derives
WO2007017242A2 (fr) * 2005-08-08 2007-02-15 Csl Behring Gmbh Nouveau procede de reduction virale
US9102762B2 (en) 2003-12-01 2015-08-11 Novo Nordisk Healthcare Ag Virus filtration of liquid factor VII compositions
US11553712B2 (en) 2010-12-30 2023-01-17 Laboratoire Français Du Fractionnement Et Des Biotechnologies Glycols as pathogen inactivating agents
US11590452B2 (en) 2017-06-12 2023-02-28 Asahi Kasei Medical Co., Ltd. Method for filtering protein-containing liquid

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0152345A2 (fr) * 1984-02-07 1985-08-21 INTERFERON SCIENCES, INC a Delaware Corporation Excipients pour l'administration d'interféron
EP0231816A2 (fr) * 1986-02-05 1987-08-12 Dr. Karl Thomae GmbH Préparations pharmaceutiques pour la stabilisation de l'interféron alpha
EP0571871A2 (fr) * 1992-05-26 1993-12-01 SEITZ-FILTER-WERKE Gmbh und Co. Procédé de fabrication d'une membrame hydrophile

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0152345A2 (fr) * 1984-02-07 1985-08-21 INTERFERON SCIENCES, INC a Delaware Corporation Excipients pour l'administration d'interféron
EP0231816A2 (fr) * 1986-02-05 1987-08-12 Dr. Karl Thomae GmbH Préparations pharmaceutiques pour la stabilisation de l'interféron alpha
EP0571871A2 (fr) * 1992-05-26 1993-12-01 SEITZ-FILTER-WERKE Gmbh und Co. Procédé de fabrication d'une membrame hydrophile

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7919592B2 (en) * 2002-03-15 2011-04-05 Zlb Behring Gmbh Method for separating off viruses from a protein solution by means of nanofiltration
EP1348445A1 (fr) * 2002-03-15 2003-10-01 Aventis Behring GmbH Procédé de séparation de virus d'une suspension de protéines par nanofiltration
AU2003200981B2 (en) * 2002-03-15 2008-11-20 Csl Behring Gmbh Method for separating off viruses from a protein solution by means of nanofiltration
JP4495404B2 (ja) * 2002-03-15 2010-07-07 ツェー・エス・エル・ベーリング・ゲー・エム・ベー・ハー ナノろ過によってタンパク溶液からウイルスを分離する方法
KR100998158B1 (ko) * 2002-03-15 2010-12-06 체에스엘 베링 게엠베하 나노여과에 의해서 단백질 용액으로부터 바이러스를 분리제거하는 방법
JP2003274941A (ja) * 2002-03-15 2003-09-30 Aventis Behring Gmbh ナノろ過によってタンパク溶液からウイルスを分離する方法
WO2005039652A2 (fr) * 2003-10-09 2005-05-06 Prodotti Chimici E Alimentari Spa Procede permettant d'eliminer les agents infectieux de l'encephalopathie spongiforme transmissible (tse) de la bile et de ses derives
WO2005039652A3 (fr) * 2003-10-09 2005-08-11 Prodotti Chimici Alimentari Procede permettant d'eliminer les agents infectieux de l'encephalopathie spongiforme transmissible (tse) de la bile et de ses derives
US9102762B2 (en) 2003-12-01 2015-08-11 Novo Nordisk Healthcare Ag Virus filtration of liquid factor VII compositions
WO2007017242A2 (fr) * 2005-08-08 2007-02-15 Csl Behring Gmbh Nouveau procede de reduction virale
WO2007017242A3 (fr) * 2005-08-08 2007-04-26 Zlb Behring Gmbh Nouveau procede de reduction virale
US11553712B2 (en) 2010-12-30 2023-01-17 Laboratoire Français Du Fractionnement Et Des Biotechnologies Glycols as pathogen inactivating agents
US11590452B2 (en) 2017-06-12 2023-02-28 Asahi Kasei Medical Co., Ltd. Method for filtering protein-containing liquid

Also Published As

Publication number Publication date
FI981337A0 (fi) 1998-06-10
FI981337A (fi) 1999-12-11
AU4783499A (en) 1999-12-30
EP1086120A1 (fr) 2001-03-28
FI106465B (fi) 2001-02-15

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