WO1991009940A1 - Formulation stable de la neuraminidase virale - Google Patents

Formulation stable de la neuraminidase virale Download PDF

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Publication number
WO1991009940A1
WO1991009940A1 PCT/US1990/007680 US9007680W WO9109940A1 WO 1991009940 A1 WO1991009940 A1 WO 1991009940A1 US 9007680 W US9007680 W US 9007680W WO 9109940 A1 WO9109940 A1 WO 9109940A1
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WIPO (PCT)
Prior art keywords
formulation
acid
virus
tris
bis
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Application number
PCT/US1990/007680
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English (en)
Inventor
James F. Maher
C. Worth Clinkscales
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Symex Corp.
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Publication date
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Publication of WO1991009940A1 publication Critical patent/WO1991009940A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16211—Influenzavirus B, i.e. influenza B virus
    • C12N2760/16251—Methods of production or purification of viral material

Definitions

  • This invention relates to a stable, aqueous formulation of enzymatically active viral neuraminidase.
  • Neuraminidase is an enzyme found in many viruses whose role has been postulated to be involved in spread of the virus from cell to cell. Some of the viruses containing this neuraminidase activity include the influenza viruses (types A and B) and the parainfluenza viruses (types 1, 2 and 3). The current assays for this enzyme have employed numerous methods (see Santer et al. Bioch. Biophys. Acta 523:435 (1978); Tuppy et al. FEBS Letters, 2:72 (1969); Myers et al. Anal. Biochem. 101:166 (1980); Warner et al. Biochem. 18:2783 (1979); Potier et al. Anal. Biochem.
  • Influenza neuraminidase is thermolabile (see Cabezas et al. Bioch. Biophys. Acta 616:228 (1980)) with the activity being reduced 60% within 10 minutes at 50°C
  • Other studies have examined the effect of several detergents such as Triton X-100, Tween 20, sodium dodecyl sulfate, NP-40, N-laurosylsarcosine, salts and cofactors on activity (see Thomas et al. Anal. Biochem. 88:461 (1978); Aitken Eur. J. Biochem. 107:51 (1980); Bottex et al. Lyon Pharm. 27:327 (1976); Bucher et al.
  • the invention is an aqueous formulation having viral neuraminidase activity comprising:
  • the formulation also includes a sufficient amount of a nonionic detergent to facilitate release of neuraminidase from the viral envelope.
  • a method of stabilizing neuraminidase activity in an aqueous formulation containing a virus with neuraminidase activity comprising adding to the formulation a stabilizing amount of a stabilizer selected from the group consisting of polyhydric sugar alcohols, simple sugars and disaccharide sugars.
  • Influenza virus and other neuraminidase- containing viruses and their methods of preparation are well known.
  • Hoyle, L. The Influenza Viruses (S. Gard, C. Hallaver and K.F. Meyer, ed.), 32-38, Springer-Verlag, New York (1968); D.J.S. Arora et al. Can. J. Microbiol. 19, 633 (1973); T. Barrett and S.C. Inglis Virology, A Practical Approach (B.W.J. Mahy, ed.), 140, IRL Press, Washington, D.C (1985); and P. Payment and M. Trudel Biotechnology Applications and Research (P.N. Cheremisinoff and R.P.
  • influenza virus and other neuraminidase containing viruses include the influenza viruses (types A and B) and the parainfluenza viruses (types 1, 2 and 3).
  • the amount of virus in the formulation will depend upon the source of virus and the intended purpose of the formulation.
  • the characteristics of the source of virus e.g., a culture of virus having a known activity and viral concentration
  • the intended purpose of the formulation is to provide a standard for neuraminidase
  • the amount of virus will be sufficient to provide a level of activity higher than the sensitivity level of the assay.
  • the characteristics of the source of virus are not known (e.g., a clinical sample of a pharyngeal, nasopharyngeal or respiratory secretion)
  • the intended purpose of the formulation is to provide a specimen for diagnosis of influenza
  • the amount of virus will be initially unknown.
  • polyhydric sugar alcohols examples include trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol, the simple sugars glucose and fructose and the disaccharide sucrose.
  • trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol
  • simple sugars glucose and fructose examples of the polyhydric sugar alcohols and simple and disaccharide sugars can be used alone or in a combination.
  • the sugar stabilizer is added to the formulation in an amount from 0.2 M to 2.1 M, preferably, 0.6 M to 2.0 M.
  • the organic and inorganic acid buffers to be used in the present invention maintain the pH of the formulation in the range of about 4.0 to 7.0, preferably 5.5 to 6.5, and can be conventional buffers of organic acids and salts thereof such as citrate buffers (e.g. monosodium citrate-disodium citrate mixture, citric acid- trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g. succinic acid- monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g.
  • citrate buffers e.g. monosodium citrate-disodium citrate mixture, citric acid- trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.
  • succinate buffers e.g. succinic acid- monosodium succinate mixture, succinic acid-sodium hydro
  • tartaric acid- tartrate mixture tartaric acid- potassium tartrate mixture, tartaric acid-sodium hydroxide mixture etc.
  • fumarate buffers e.g. fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate acid-disodium fumarate mixture
  • gluconate buffers e.g. gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.
  • oxalate buffers e.g.
  • oxalic acid-sodium oxalate mixture oxalic acid- sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.
  • lactate buffers e.g. lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.
  • acetate buffers e.g.
  • acetic acid-sodium acetate mixture acetic acid-sodium hydroxide mixture, etc.
  • phosphate buffers e.g., monosodium phosphate-disodium phosphate mixture, monosodium phosphate-sodium hydroxide mixture, trisodium phosphate-hydrochloric acid mixture, etc.
  • 2-(N-morpho- lino)ethanesulfonc acid [bis-(2-hydroxy- ethyl)imino]tris (hydroxymethyl)methane, N-2-acetamidoiminodiacetic acid, 1,3-bis[tris(hydroxymethyl)methylamino]propane, piperazine-N,N'-bis(2-ethanesulfonic acid), N-2-acetamido-2-aminoethanesulfonic acid, 3-(N-morpholino)-2-hydroxypropanesulfonic acid, N-N-bis-(2-hydroxyethyl)2-aminoe
  • nonionic detergents examples include the Pluronics, for example, Polysorbate 80 or Polysorbate 20, Triton X-100, NP-40 and the alkyl glucosides, for example, octyl glucoside and nonyl glucoside. When used, these detergents are present in the range of 0.1 to 10.0% by weight with a preferred range of about 1.0 to 7.0% by weight.
  • Examples of a divalent metal to be added is calcium ion.
  • the ion may be added in the form of water soluble salts. It should be added at a level between 1 and 20 mM, preferably at 10 mM. There are some indications that this divalent cation is required for the activity of some of the neuraminidases found in viruses and therefore is included to assure maximal activity of the neuraminidase within the viruses (see S.M. Carroll and
  • the aqueous formulation of this invention is stable for prolonged periods of time.
  • the formulation of this invention may be stored in a liquid state at various temperatures.
  • a preferred storage temperature range is between about 2° and about 8°C.
  • Example 1 Influenza virus type A was put in the liquid formulation/excipient system after growth in a cell culture system and initial purification through clarification of the cell culture media. It was put at a level 10 - 100 times the sensitivity of a modified fluorescent assay procedure (see Kiyotani and associates Zbl. Bakt. Hyg. A. 260, 273, (1985); and T.G. Warner and J-S. O'Brien Biochemistry 18, 2783 (1979)) into: 50 mM acetic acid-sodium hydroxide mixture; 10 mM CaCl 2 , 0.6 to 2.1 M of stabilizer (as shown in Tables 1 and 2), and a sufficient quantity of deionized water. The acetate buffer maintained the pH at 5.9. This liquid formulation/ excipient system was found to exhibit an increased stability at 40°C over the length of the study when compared to the same level of influenza A virus without the added stabilizer.
  • the loss of neuraminidase activity reflected in the rate constants indicates an approximate 4- to 30-fold increased level of stabilization over the control Influenza A virus with no added stabilizer. These changes are reflected in the rate constant which is the slope of the line resulting from the plot of the logarithm of the loss of neuraminidase activity of the liquid formulation/excipient system versus time. Enzyme activity was measured using the general fluorometric procedure described by Kiyotani et al., supra. The stability of the liquid formulation/excipient system was considerably greater than that of the control virus without the added stabilizer.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Formulations aqueuses de virus à activité de neuraminidase, par exemple virus grippaux et para-grippaux. Elles sont rendues thermostables par l'adjonction d'un stabilisant du sucre, d'un tampon de pH 4-7, d'un ion calcium, et éventuellement, d'un détergent non ionique.
PCT/US1990/007680 1989-12-29 1990-12-27 Formulation stable de la neuraminidase virale WO1991009940A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US459,064 1983-01-18
US45906489A 1989-12-29 1989-12-29

Publications (1)

Publication Number Publication Date
WO1991009940A1 true WO1991009940A1 (fr) 1991-07-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2358387A1 (fr) * 2008-11-14 2011-08-24 Baxter International Inc. Formulations de vaccin et leurs utilisations

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3927209A (en) * 1973-05-11 1975-12-16 Bayer Ag Para-influenza-3-virus
US3961046A (en) * 1974-12-02 1976-06-01 American Cyanamid Company Mumps vaccine and preparation thereof
US4147772A (en) * 1976-02-03 1979-04-03 Merck & Co., Inc. Vaccine stabilizer
US4356169A (en) * 1980-04-14 1982-10-26 Europaisches Laboratorium Fur Method of preparing an immunogenic membrane protein aggregate of influenza and parainfluenza viruses and rhabdoviruses
US4537769A (en) * 1982-04-06 1985-08-27 American Cyanamid Company Stabilization of influenza virus vaccine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3927209A (en) * 1973-05-11 1975-12-16 Bayer Ag Para-influenza-3-virus
US3961046A (en) * 1974-12-02 1976-06-01 American Cyanamid Company Mumps vaccine and preparation thereof
US4147772A (en) * 1976-02-03 1979-04-03 Merck & Co., Inc. Vaccine stabilizer
US4356169A (en) * 1980-04-14 1982-10-26 Europaisches Laboratorium Fur Method of preparing an immunogenic membrane protein aggregate of influenza and parainfluenza viruses and rhabdoviruses
US4537769A (en) * 1982-04-06 1985-08-27 American Cyanamid Company Stabilization of influenza virus vaccine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICA ET BIOPHYSICA ACTA, Vol. 616, issued 1980, CABEZAS et al., "Neuraminidase from Influenza Virus A (H3N2). Specificity Towards Several Substrates and Procedure of Activity Determination", page 228-238. *
BIOLOGICAL ABSTRACTS, Volume 63, No. 7, issued 01 April 1977, BAKER et al., "Effect of CA# on the Stability of Influenza Virus Neuraminidase", see pages 4163-4164, Abstract No. 42287; & ARCH. VIROL., 52(1/2), 7-18. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2358387A1 (fr) * 2008-11-14 2011-08-24 Baxter International Inc. Formulations de vaccin et leurs utilisations
US8795683B2 (en) 2008-11-14 2014-08-05 Baxter International Inc. Vaccine formulations and uses thereof
EP2358387B1 (fr) * 2008-11-14 2017-04-19 Nanotherapeutics, Inc. Formulations de vaccin et leurs utilisations

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AU7161591A (en) 1991-07-24

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