EP0702690A1 - Compositions lyophilisees comprenant de l'arn - Google Patents

Compositions lyophilisees comprenant de l'arn

Info

Publication number
EP0702690A1
EP0702690A1 EP95915190A EP95915190A EP0702690A1 EP 0702690 A1 EP0702690 A1 EP 0702690A1 EP 95915190 A EP95915190 A EP 95915190A EP 95915190 A EP95915190 A EP 95915190A EP 0702690 A1 EP0702690 A1 EP 0702690A1
Authority
EP
European Patent Office
Prior art keywords
rna
freeze
dried
nucleic acid
particles
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Withdrawn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
EP95915190A
Other languages
German (de)
English (en)
Inventor
Marcelus Hendrikus Franciscus Uylen
Franciscus Henricus Maria Van Dinther
Tim Kievits
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.)
Akzo Nobel NV
Original Assignee
Akzo Nobel NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Akzo Nobel NV filed Critical Akzo Nobel NV
Priority to EP95915190A priority Critical patent/EP0702690A1/fr
Publication of EP0702690A1 publication Critical patent/EP0702690A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • the invention relates to freeze-dried compositions comprising ribonucleic acid (RNA) and methods for the preparation of said compositions.
  • the invention further relates to the use of said compositions in methods for the detection of nucleic acid as well as test-kits for the detection of nucleic acid comprising said compositions.
  • RNA is known to be a rather sensitive and relatively unstable entity. RNA in solution is subject to more or less rapid deterioration. Especially degradation by RNase is a major concern when handling RNA preparations. This omnipresent enzyme (RNase is present on fingertips, in dust etc.) is heat stable and does not require cofactors for its enzymatic activity. This makes it difficult to inactivate RNase in RNA preparations. Although there are several isolation methods yielding RNase-free RNA (Sambrook J., Fritch E.F. and Maniatis T. (1989) Moecular Cloning: A Laboratory Manual, 2nd edn Vol.1 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor; Ausubel, F.M. et al.
  • RNA can also be solubilized in formamide instead of DEPC-treated water.
  • Formamide effectively protects RNA from degradation by RNase, and allows for long-term storage at -20°C.
  • the present invention provides freeze dried RNA-containing particles.
  • freeze-drying process has no negative effect on the RNA and substantially retards all possible inactivation processes.
  • RNA-containing compositions according to the invention are therefore very useful alternatives for, for example, presently used RNA-containing reagent solutions.
  • the particles according to the present invention are dry and therefore almost insensitive to various degradation processes.
  • RNA sequences of different lenghts can be freeze-dried. All RNA sequences varying in length from, for example, a few hundred up to thousands of nucleotides can be incorporated in the freeze-dried compositions according to the present invention.
  • freeze-dried particles according to the present invention can be presented in different paniculate forms.
  • RNA in these freeze-dried particles is stable during storage for at least one year at 2-8°C up to 37°C.
  • the RNA is freeze-dried in the form of a spherical particle.
  • Spherical freeze-dried particles (accuspheres or lyospheres) are known from Price et al. (US 3,655,838). These spherical beads, contain material for immunological reactions.
  • Lyospheres comprising biological active materials are known, for instance from USP 3,932,943, and also from many other patents. Advantages of lyospheres are the uniformity of the particles, the easy to handle products, the faster freeze- dry process, less degradation during the freeze-dry process, and the improved dissolution properties.
  • the lyospheres can be prepared by freeze-drying droplets of an aqueous RNA- solution.
  • the solution can be sprayed into a cold bath (for example as disclosed in DT 2,140,747, EP 8191 3, or US 3,928,566), into liquid nitrogen (for example as disclosed in J5 91 69,504), or onto a refrigerated drum (for example as disclosed in USP 3892876) or a refrigerated plate (for example as disclosed in US 4,501 ,719).
  • a cold bath for example as disclosed in DT 2,140,747, EP 8191 3, or US 3,928,566
  • liquid nitrogen for example as disclosed in J5 91 69,504
  • a refrigerated drum for example as disclosed in USP 3892876
  • a refrigerated plate for example as disclosed in US 4,501 ,719
  • the freeze dried particles according to the present invention may comprise, in addition to the RNA, stabilizers such as polyhydroxy-compounds like sucrose, mannitol, dextran, sorbitol and glycerol.
  • stabilizers such as polyhydroxy-compounds like sucrose, mannitol, dextran, sorbitol and glycerol.
  • the freeze-dried particles according to the present invention are especially useful as diagnostic reagents in methods for the detection of nucleic acid. Diagnostic methods for the detection of nucleic acid usually involve the amplification of nucleic acid, isolated from a clinical sample.
  • NASBA nucleic acid sequence based amplification
  • an internal control sequence is sometimes added to the sample.
  • An internal control sequence as used with the method described in PCT/EP93/02248 comprises a nucleic acid sequence distinguishable from the target sequence that can be amplified with the same amplification reagents as the target sequence.
  • freeze-dried particles can be used.
  • a method for the quantification of nucleic acid, using the addition of a known number of molecules of a nucleic acid sequence, corresponding to the target nucleic acid, to a sample containing an unknown amount of the target nucleic acid sequence has been described in co-pending co-owned European patent application published under no. EP 525882.
  • the method as described in EP 525882 is based on the principle of amplification of nucleic acid from a sample containing an unknown concentration of wild-type target nucleic acid, to which has been added a known amount of a well-defined mutant sequence. Amplification is performed with one primer set capable of hybridizing to the target as well as the mutant sequence.
  • the competitive amplification described in EP 525882 can be performed with a fixed amount of sample and dilution series of mutant sequence or vice versa.
  • the mutant sequence can again be incorporated in a freeze-dried particle, which might also contain the primers and probes needed for carrying out the quantitative method as described above.
  • freeze-dried particles according to the present invention are preferably produced in the form of spheres.
  • Spherical particles according to the invention are preferably produced by a method comprising the steps of preparing an RNA solution comprising one or more stabilizers and forming freez-dryed particles form said solution, preferably by dripping said solution into liquid nitrogen, keeping the droplets in the liquid nitrogen until they are completely frozen and lyophilizing said droplets.
  • freeze-dried particles according to the present invention can be incorporated in a test-kit for the detection or quantification of nucleic acid, together with other reagents used with the detection method employed, such as suitable enzymes, primers and probes.
  • Example 1 Preparation of RNA-containing freeze-dried particles.
  • a stabilizer mixture was prepared using 15% w/w saccharose, 5% w/w mannitol and 5% w/w dextran T-40 in water.
  • the saccharose was obtained from Sigma, mannitol from Baker and Dextran T40 from Farmacia).
  • RNA solution was prepared by 100 times dilution in DEPC- treated water of a concentrated RNA stock. 50 ml of the stabilizer solution was mixed with 50 ml of the RNA solution.
  • Lyospheres were prepared from this RNA/stabilizer solution in the following way:
  • RNA/stabilizer mixture 50 ⁇ portions of the RNA/stabilizer mixture were dripped into boiling nitrogen. The spheres were kept into liquid nitrogen until they were completely frozen. The frozen spheres were lyophilized yielding spheres with a residual moisture content below 2% w/w and essentially having the same diameter as the frozen spheres. Each accusphere thus prepared contained 1.22 # 10 7 RNA molecules.
  • RNA-containing freeze-dried particles can be prepared in the following way:
  • RNA solutions were prepared for the preparation of 600 accuspheres containing 10 ⁇ 2 molecules of each species of RNA per accusphere.
  • the solution consisted of three different RNA species each in a volume of 9ml (total volume of 27ml).
  • the RNA solutions contained 1 ,5 M sodiumchloride, sodiumchloride was obtained from Janssen Chimica.
  • the same stabilizers as used under A were added to the RNA mixture as dry components.
  • 2.25 g of saccharose, 0.75 g of mannitol and 0.75 g of dextran T40 was added to a tube containing the RNA mixture. Water was added to the RNA/stabilizer mixture until a final volume of 30 ml was obtained.
  • Freeze-dried particles containing RNA can likewise be prepared in the following way:
  • RNA solution was prepared for the preparation of 600 accuspheres containing 7.6* 10 ⁇ RNA molecules per accusphere.
  • RNA molecules per ⁇ was added to 30 ml of the sucrose/mannitol/dextran
  • Example 2 Stability of freeze-dried particles comprising RNA.
  • RNA as a freeze-dried sphere was tested by comparing the amplification efficiency of NASBA reactions using RNA from lyophilized spheres according to the invention and an independend reference.
  • the lyophilized spheres were prepared as described under "A" in example 1 .
  • the reference Wild-type (WT) is an in vitro generated HIV-1 cell line quantitated by means of electron microscopy as 2.9 ( ⁇ 1 .6) x10 A 10 viral particles per ml (layne et al., 1992). This reference was diluted in lysis buffer and stored as single use aliquots of 100 //I as 7300 RNA molecules per ⁇ l at -70°C.
  • RNA spheres tested in this stability study were four different batches of RNA spheres, stored at 2-8°C, 20-25 °C and 35-38°C.
  • RNA sphere was diluted in 550 / I of elution buffer (1 mmol/l Tris/HCI pH 8.5), concentration of the three RNAs, Qa, Qb and Qc respectively 1000000, 100000 and 0000 RNA molecules per 50 ⁇ l of elution buffer.
  • Qa, Qb and Qc are in vitro synthesized RNA sequences having a length of approximately 1000-1500 nucleotides.
  • An aliquot of 50 ⁇ of the dissolved RNA sphere was added to the lysis buffer tube and mixed.
  • Detection of HIV-1 RNA in a sample is based on the electrochemi-luminescence principle (Blackburn GF, Shah HP, Kenten JH, Leland J, Kamin RA, Link J, Peterman J, Powell MJ, Shah A, Talley DB et al: Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics, Clin Chem 1991 ; 37:1534-1 539, Kenten JH, Gudibande S, Link J, Willey JJ, Curfman B, Major EO, Massey RJ: Improved electrochemiluminescent label for DNA probe assays: rapid quantitative assays of HIV-1 polymerase chain reaction products, Clin Chem 1992; 38:873-879).
  • amplificates WT, Qa, Qb and Qc
  • aliquots of the amplified sample were added to four hybridization solutions, each specific for one of the amplificates.
  • the respective amplificates were hybridized with a bead-oligo (i.e. a biotin- oligo bound to streptavidin coated magnetic beads acting as the solid phase) and a ruthenium-labeled probe.
  • the magnetic beads carrying the hybridized amplificate/probe complex were captured on the surface of an electrode by means of a magnet. Voltage applied to this electrode triggers the electrochemiluminescence (ECL) reaction.
  • ECL electrochemiluminescence
  • the light emitted by the hybridized ruthenium-labeled probes is proportional to the amount of amplificate. Calculation based on the relative amounts of the four amplificates reveals the amount of WT reference.
  • NASBA assay can be observed after storage of the RNA spheres up to 14 weeks at all tested temperatures (table 1 ).
  • RNA sequences of approximately 1500 nucleotides, prepared according to the protocol C of example 1 were dissolved in 200 //I elution buffer (10 mM Tris/HCI, pH 8.5). 20 l of this solution was added to 9ml lysis buffer (5 M guanidin thiocyanate, 1 % (v/v) Triton X-100, 20mM EDTA, 50 mM Tris/HCI, pH 6.4). Subsequently 70 ⁇ activated silica (1 mg/ml size selected suspension in 0.1 N HCI) was added to the lysis mixture to bind the nucleic acid. After washing and drying the silica, nucleic acid was eluted in 100 ⁇ elution buffer.
  • the final volume of the reaction mixture is 25 ⁇ , containing 40 mM Tris/HCI, pH 8.5, 12mM MgCl2, 40 mM KCI, 5mM DTT, 1 mM of each dNTP, 2 mM of each NTP, 15% (v/) DMSO, 0.1 / g/ ⁇ l BSA, 0.2 //I of each of the oligonucleotide primers, 8.0 u AMV-RT, 0.1 u RNase-H and 40.0 u T7 RNA Polymerase.
  • One primer carries a T7 RNA polymerase recognition site.
  • 8 ⁇ of the isolated RNA was used (containing approximately 200 molecules of RNA).
  • the amplification reactions are performed in centrifuge micro test tubes at 41 °C for 90 minutes.
  • the amplifcation products are analysed by enzyme-linked gel assay (ELGA).
  • Amplified RNA is detected by a non-radioactive hybridization in solution, using sequence specific HRP ⁇ '-labelled oligonucleotide probes. After hybridization, non-hybridized probes were separated form the homologeous hybridized product by gel eiectrophoresis. Free HRP probes and hybridized products were directly visualized in a polyacrlyiamide gel by incubating the gel in a substrate solution for HRP.
  • RNA accusphere lots were analyzed as described above, in a long-term stability study.
  • the first two batches show no sign of RNA degradation upon storage of the gelmarker RNA accuspheres at 4°C for 52 weeks.
  • the third batch was tested after 36 weeks and also showed the expected gel pattern without degradation. If degradation of RNA had occurred this would have been indicated by the appearance of RNA smears in the gel instead of the discrete bands that were found.

Abstract

Particules lyophilisées comprenant de l'acide ribonucléique (ARN) et leur procédé de préparation. L'invention se rapporte en outre à l'utilisation de ces particules dans des procédés de détection de l'acide nucléique, de même que dans des tests prêts à l'emploi de détection de l'acide nucléique comprenant ces particules. En lyophilisant l'ARN, on obtient un produit stable que l'on peut conserver pendant une longue période de temps sans que ce produit perde sa structure. En conséquence, les particules lyophilisées de l'invention, qui comprennent l'ARN, constituent des alternatives très utiles destinées, par exemple, aux solutions actuellement utilisées de réactifs contenant de l'ARN. Les particules de l'invention sont sèches et, par conséquent, pratiquement insensibles aux divers processus de dégradation.
EP95915190A 1994-04-07 1995-04-07 Compositions lyophilisees comprenant de l'arn Withdrawn EP0702690A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP95915190A EP0702690A1 (fr) 1994-04-07 1995-04-07 Compositions lyophilisees comprenant de l'arn

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP94200953 1994-04-07
EP94200953 1994-04-07
PCT/EP1995/001268 WO1995027721A1 (fr) 1994-04-07 1995-04-07 Compositions lyophilisees comprenant de l'arn
EP95915190A EP0702690A1 (fr) 1994-04-07 1995-04-07 Compositions lyophilisees comprenant de l'arn

Publications (1)

Publication Number Publication Date
EP0702690A1 true EP0702690A1 (fr) 1996-03-27

Family

ID=8216778

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95915190A Withdrawn EP0702690A1 (fr) 1994-04-07 1995-04-07 Compositions lyophilisees comprenant de l'arn

Country Status (4)

Country Link
EP (1) EP0702690A1 (fr)
JP (1) JPH08511956A (fr)
AU (1) AU2215995A (fr)
WO (1) WO1995027721A1 (fr)

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US7276359B1 (en) 1998-03-13 2007-10-02 Wyeth Polynucleotide composition, method of preparation, and use thereof
EP1555033A3 (fr) * 1998-03-13 2005-08-17 Wyeth Composition polynucléotidique, procédé de préparation et utilisation
EP1061955B1 (fr) * 1998-03-13 2005-05-04 Wyeth Composition polynucleotidique, procede de preparation et utilisation
ES2470340T3 (es) 2005-12-28 2014-06-23 Advanced Bionutrition Corporation Vehículo de administración para bacterias probi�ticas que comprende una matriz seca de polisac�ridos, sac�ridos y polioles en forma vítrea
KR100777249B1 (ko) 2006-02-14 2007-11-28 (주)바이오니아 건조 올리고뉴클레오티드 조성물 및 이의 제조 방법
JP5285617B2 (ja) 2006-12-18 2013-09-11 アドバンスド バイオニュートリション コーポレーション 生きたプロバイオティクスを含む乾燥食物製品
EP2071034A1 (fr) 2007-12-12 2009-06-17 bioMérieux Procédé pour le traitement d'une solution en vue d'éliminer un acide ribonucléique après amplification
EP3266448B1 (fr) 2009-03-27 2022-02-16 Intervet International B.V. Vaccins microparticulaires utilisables pour procéder à une vaccination et à des rappels par voie orale ou nasale chez les animaux, dont les poissons
CN102459568A (zh) 2009-05-26 2012-05-16 先进生物营养公司 包含生物活性微生物和/或生物活性材料的稳定干粉组合物及其制造方法
CN105255881A (zh) * 2009-07-31 2016-01-20 埃泽瑞斯公司 用于蛋白质表达的具有未修饰和修饰核苷酸的组合的rna
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HUE047796T2 (hu) 2010-07-06 2020-05-28 Glaxosmithkline Biologicals Sa RNS bevitele több immunútvonal bekapcsolására
US20130171241A1 (en) 2010-07-06 2013-07-04 Novartis Ag Liposomes with lipids having an advantageous pka-value for rna delivery
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EP3928800A3 (fr) 2015-05-20 2022-03-23 CureVac AG Composition de poudre sèche comprenant de l'arn à longue chaîne
US10517827B2 (en) 2015-05-20 2019-12-31 Curevac Ag Dry powder composition comprising long-chain RNA
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Also Published As

Publication number Publication date
AU2215995A (en) 1995-10-30
WO1995027721A1 (fr) 1995-10-19
JPH08511956A (ja) 1996-12-17

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