(19) TZZ ¥_T (11) EP 2 742 983 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: B01D 15/38 (2006.01) 23.08.2017 Bulletin 2017/34 (21) Application number: 12196536.2 (22) Date of filing: 11.12.2012 (54) Method for isolating fragrance and flavour compounds Verfahren zum Isolieren von Duft- und Geschmackskomponenten Procédé permettant d’isoler des composés d’arôme et de parfum (84) Designated Contracting States: (56) References cited: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB WO-A1-2012/172546 GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR • BHATNAGAR R ET AL: "SEPARATION OF NONTARGET COMPOUNDS BY DNA (43) Date of publication of application: APTAMERS", ANALYTICAL CHEMISTRY, 18.06.2014 Bulletin 2014/25 AMERICAN CHEMICAL SOCIETY, US, vol. 72, no. 4, 15 February 2000 (2000-02-15), pages 827-831, (73) Proprietor: Symrise AG XP000924024, ISSN: 0003-2700, DOI: 37603 Holzminden (DE) 10.1021/AC991112F • RAVELET C ET AL: "Liquid chromatography, (72) Inventors: electrochromatography and capillary • Krammer, Gerhard electrophoresis applications of DNA and RNA 37603 Holzminden (DE) aptamers", JOURNAL OF CHROMATOGRAPHY, • Degenhardt, Andreas ELSEVIER SCIENCE PUBLISHERS B.V, NL, vol. 37603 Holzminden (DE) 1117, no. 1, 2 June 2006 (2006-06-02) , pages 1-10, • Walter, Johanna-Gabriela XP024967352, ISSN: 0021-9673, DOI: 31582 Nienburg (DE) 10.1016/J.CHROMA.2006.03.101 [retrieved on • Scheper, Thomas 2006-06-02] 30559 Hannover (DE) • JOHANNA-GABRIELA WALTER ET AL: • Stahl, Frank "Aptamers as affinity ligands for downstream 30419 Hannover (DE) processing", ENGINEERING IN LIFE SCIENCES, vol. 12, no. 5, 25 June 2012 (2012-06-25), pages (74) Representative: Fabry, Bernd 496-506, XP055058513, ISSN: 1618-0240, DOI: IP2 Patentanwalts GmbH 10.1002/elsc.201100197 Schlossstrasse 523 • ERIC PEYRIN: "Nucleic acid aptamer molecular 41238 Mönchengladbach (DE) recognition principles and application in liquid chromatography and capillary electrophoresis", JOURNAL OF SEPARATION SCIENCE, vol. 32, no. 10, 15 April 2009 (2009-04-15), pages 1531-1536, XP055058515, ISSN: 1615-9306, DOI: 10.1002/jssc.200900061 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 742 983 B1 Printed by Jouve, 75001 PARIS (FR) 1 EP 2 742 983 B1 2 Description be isolated or, respectively, towards one, more or all of the target compounds to be isolated, [0001] The present invention relates to a method for wherein the aptamer(s) is/are immobilized, prefera- isolating target flavor compounds or fragrance com- bly covalently immobilized, on a support, the support pounds on a preparative scale using specific aptamers 5 being preferably selected from the group consisting immobilized on a support (as described herein below). of magnetic particles, organic polymers, inorganic [0002] Flavor and fragrance compounds continue to polymers, bio-polymers, membrane adsorbers and be of significant commercial relevance. Especially cos- monolithic materials, metics and food industries are on a constant search for suitable substances and efficient methods to isolate or 10 (iii) mixing the aptamer(s) with the composition con- produce them. Extraction from natural sources as well sisting of the target compound(s) to be isolated and as synthetic or semi-synthetic access to compounds nor- one or more further compounds, and specifically mally requires a selective isolation of the target molecule binding the compound(s) to be isolated to the aptam- from complex mixtures such as extracts or reaction mix- er(s), tures. Typical purification methods known in the prior art 15 include solid phase extraction using polymers, liquid/liq- (iv) optionally, removing one, more or all compounds uid partitioning by solubility, membrane separation by of the composition which are not specifically bound size exclusion or pervaporation. to the aptamer(s) and/or removing (other) impurities, [0003] Isolation methods which are based on intermo- and lecular recognition developed by an evolutionary selec- 20 tion process such as antigen/antibody interactions are (v) releasing the bound target compound(s) from the also believed to be efficient in terms of specificity and aptamer(s). therefore well suited for analytical purposes. Their large(r)-scale applicability, however, often suffers from a [0006] Specific molecular recognition by nucleic acid loss in performance and/or the limited availability of spe- 25 motifs has gained a wide therapeutic and analytic scope. cific capture agents. This is especially true for antibodies, Aptamer sequences are used as biosensors, for example which are produced in cell culture and therefore require on biosensor arrays for the detection of various analytes large expenses of time and cost for the purification from and the removal of contaminants (e.g. bisphenol A, food the fermentation broth and are prone to large batch-to- borne pathogens such as Campylobacter, Listeria, Es- batch variations. Moreover, antibodies are generated by 30 cherichia, Staphylococcus or Clostridium). The use of immunization which is not applicable for all target com- nucleic acid aptamers has several distinct advantages pounds (e.g. toxic or non-immunogenic substances), and over the introductory mentioned antigen/antibody sys- their efficient use is restricted to conditions close to the tems which mainly arise from their preparation by a com- physiological range as they are highly susceptible to binatorial chemistry approach. In-vitro methods like SE- changes in pH and temperature. 35 LEX (systematic evolution of ligands by exponential en- [0004] Thus, there is still a need for an optimized proc- richment, Ellington and Szostak, Nature, 1990, 346, ess for selectively isolating relevant target molecules 818-822; Robertson and Joyce, Nature, 1990, 344, such as flavor or fragrance compounds from complex 467-468; Tuerk and Gold, Science, 1990, 249, 505-510) mixtures, preferably in a preparative scale. or Monolex (Nitsche et al., BMC Biotechnol., 2007, 7, 48) [0005] The present invention now provides a novel, 40 are not restricted by biological/physiological limitations, particularly advantageous method for isolating, prefera- which means that aptamers can advantageously be se- bly in preparative scale, one or more target compounds, lected against virtually any kind of target ligands and for wherein the or, respectively, one, more or all of the target use under virtually any desired conditions (e.g. regarding compounds are selected from the group consisting of pH or temperature). Oligonucleotide libraries are pro- flavor compounds and fragrance compounds, in partic- 45 duced synthetically and the sequence(s) with the highest ular compounds having one, more or all of the taste im- affinity to the target compound is/are identified in an it- pressions sweet, umami, warming and tingling, and com- erative process under increasingly stringent binding con- pounds having a bitter and/or an astringent taste impres- ditions. Furthermore, aptamers are very stable against sion, and compounds with (flavor and/or fragrance) mod- pH and temperature extremes and can be regenerated ifying properties, 50 without loss of binding activity. comprising the following steps: [0007] Various therapeutic and analytic methods have been developed in the prior art, which are based on the (i) providing a composition consisting of the target specific ligand recognition by aptamer sequences. compound(s) to be isolated and one or more further [0008] For example, in a review in Applied Microbiol- compounds, 55 ogy and Biotechnology, 2005, 69, 367-374, Proske et al. summarize recent developments in aptamer technology (ii) providing one or more aptamers having specific and validate their suitability as, for example, diagnostic binding capability towards the target compound to tools. 2 3 EP 2 742 983 B1 4 [0009] Tombelli et al. describe in Proceedings of SPIE aration of nontarget compounds by DNA aptamers"; - The International Society for Optical Engineering, 2007, Anal. Chem., Vol.72(4), 827-831) also disclose the sep- 6585, analytical applications of aptamers and their po- aration of small molecules, namely amino-acids enanti- tential as tools for diagnostics and in environmental and omers and polycyclic aromatic hydrocarbons, using food analysis. 5 aptamers attached on a capillary. [0010] The use of aptamers in liquid chromatography, [0020] The cited prior art demonstrates the high ver- capillary electrophoresis and electrochromatography is satility of aptamer technology in biochemical methods. for example discussed by Ravelet et al. in J Chromatogr However, the above listed applications have been de- A, 2006, 1117, 1-10. signed and optimized for small-scale use (only), which [0011] A method for microaffinity purification of pro- 10 is automatically implied in analytical, diagnostic and ther- teins using photo-cleavable RNA aptamers on affinity apeutic uses. beads was proposed by Chung et al. in Electrophoresis, [0021] The inventors now found out that besides diag- 2005, 26, 694-702, and found useful to identify target nostic and therapeutic analytes and agents, aptamers proteins from a protein mixture for a potential