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Tepzz 4 748¥B T (19) TZZ ¥_T (11) EP 2 427 483 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 14/435 (2006.01) A01N 1/02 (2006.01) 11.03.2015 Bulletin 2015/11 C12N 5/00 (2006.01) A61K 38/02 (2006.01) (21) Application number: 10721419.9 (86) International application number: PCT/EP2010/056299 (22) Date of filing: 07.05.2010 (87) International publication number: WO 2010/128159 (11.11.2010 Gazette 2010/45) (54) NOVEL HEAMOGLOBIN AND USES THEREOF NEUARTIGES HÄMOGLOBIN UND DESSEN VERWENDUNGEN Nouvelle hémoglobine et ses utilisations (84) Designated Contracting States: • ECONOMIDES A P ET AL: "The respiratory AL AT BE BG CH CY CZ DE DK EE ES FI FR GB function of the blood of Neanthes ( = Nereis) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO virens (Sars) (Polychaeta: Nereidae)" PL PT RO SE SI SK SM TR COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY 1975, vol. 51, no. 1 A, 1975, pages (30) Priority: 07.05.2009 US 176271 P 219-223, XP023590958 ISSN: 0010-406X 07.05.2009 EP 09305415 • SUZUKI T ET AL: "The giant extracellular hemoglobin from the polychaete Neanthes (43) Date of publication of application: diversicolor. The cDNA-derived amino acid 14.03.2012 Bulletin 2012/11 sequence of linker chain L2 and the exon/ intron boundary conserved in linker genes" (73) Proprietor: Hemarina BIOCHIMICA ET BIOPHYSICA ACTA - GENE 29600 Morlaix (FR) STRUCTURE AND EXPRESSION 1994 NL, vol. 1217, no. 3, 1994, pages 291-296, XP023469023 (72) Inventors: ISSN: 0167-4781 • ROUSSELOT, Morgane • CHRISTINE CHABASSE ET AL: "Gene Structure F-29420 Plouenan (FR) and Molecular Phylogeny of the Linker Chains • DUTHEIL, Delphine from the Giant Annelid Hexagonal Bilayer F-29250 Saint Pol de Léon (FR) Hemoglobins" JOURNAL OF MOLECULAR •ZAL,Franck EVOLUTION, SPRINGER-VERLAG, NE, vol. 63, F-29679 Morlaix Cedex (FR) no. 3, 12 July 2006 (2006-07-12), pages 365-374, XP019441462 ISSN: 1432-1432 (74) Representative: Cabinet Plasseraud • WAXMAN L: "THE STRUCTURE OF ANNELID 52, rue de la Victoire AND MOLLUSK HEMO GLOBINS" JOURNAL OF 75440 Paris Cedex 09 (FR) BIOLOGICAL CHEMISTRY, vol. 250, no. 10, 1975, pages 3790-3795, XP002539399 ISSN: 0021-9258 (56) References cited: • ROUSSELOT MORGANE ET AL: "Arenicola WO-A-2009/050343 WO-A1-2010/122080 marina extracellular hemoglobin: a new DE-A1- 10 220 990 FR-A1- 2 917 292 promising blood substitute" BIOTECHNOLOGY US-A1- 2003 181 358 JOURNAL,WILEY- VCHVERLAG, WEINHEIM, DE, vol. 1, no. 3, 1 January 2006 (2006-01-01) , pages 333-345, XP002480915 ISSN: 1860-6768 cited in the application 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 2 427 483 B1 Printed by Jouve, 75001 PARIS (FR) EP 2 427 483 B1 Description FIELD OF THE INVENTION 5 [0001] The invention relates to a novel isolated haemoglobin that can serve for cell culture, and organ preservation solution. BACKGROUND OF THE INVENTION 10 [0002] The present invention relates to haemoglobin and its property to fix, carry and release oxygen in a simple partial pressure of oxygen. One particular interest of such haemoglobin is its use as oxygen carrier for industrial and therapeutic applications. Two viable categories of oxygen therapeutics currently exist: haemoglobin-based oxygen carriers (HBOCs) and perfluorocarbons (PFCs). Haemoglobin taken directly from red blood cells can not be used as an intravascular oxygen carrier. To avoid spontaneous breakdown of haemoglobin and the toxicity of haemoglobin extracted from red 15 blood cells, haemoglobin-based oxygen carriers use purified human (Riess et al. Chem Rev 2001, 101:2797), animal (bovine) (Lok et al Nature 2001, 410:855) or recombinant (Looker et al Nature 1992, 356:258) haemoglobin as raw materials. Each chain of purified haemoglobin is then covalently bridged with other globin chains or microencapsulated. Perfluorocarbons are liquid fluorinated hydrocarbon compounds capable of dissolving large amounts of oxygen and then delivering this oxygen. However, in this case the oxygen partial pressure needs to be higher than air oxygen partial 20 pressure. [0003] Oxygen therapeutics have been developed for two major purposes: i) they can function as alternatives to blood transfusion in order to avoid, reduce or delay transfusion of allogenic blood and ii) improvement of tissue oxygenation of organs with poor blood supply. [0004] The inventors have previously shown that the extracellular polymeric haemoglobin of the polyachete annelid 25 Arerzicola marina can be used as an oxygen therapeutic (Rousselot et al 2006 Biotech Journal 1:333). Said haemoglobin is easily available and can be purified to a homogeneous product avoiding costly synthetic steps. It is also easy to store and is less likely to cause immunogenic responses as it is not glycosylated as already demonstrated. Economides et al ("The respiratory function of the blood of Neanthes (=Nereis) virens (Sars) (Polychaeta:Nereidae)", Comparative Bio- chemistry and Physiology 1975, vol.51, no.1A, pages 219-223) describes the hemoglobin of Neanthes virens. 30 [0005] Suzuki et al ("The giant extracellular hemoglobin from the polychaete Neanthes diversicolor. The cDNA-derived amino acid sequence of linker chain L2 and the exon/intron boundary conserved in linker genes", Biochimica et Biophysica Acta - Gene Structure and expression 1994 NL, vol.1217, no.3, pages 291-296) discloses purified haemoglobin from Neanthes diversicolor. [0006] Chabasse et al ("Gene structure and molecular phylogeny of the linker chains from the giant Annelid hexagorzal 35 bilayer hemoglobins", Journal of molecular evolution, vol.63, no.3, 12 July 2006, pages 365-374) and Waxman ("The structure of Annelid and Mollusk hemoglobins", Journal of Biological Chemistry, vol.250, not.10, 1975, pages 3790-3795) disclose Annelid haemoglobin structures. [0007] WO2009/050343 discloses the use of Annelids’ haemoglobin in a preservation solution for organs, tissues or cells. 40 [0008] DE10220990 discloses the use of hemoglobins for the controlled delivery of oxygen to cultures of tissues, cells or organs. [0009] Rousselot et al ("Arenicola marina extracellular haemoglobin: a new promising blood substitute", Biotechnology Journal, viol.1, no.3, 1 January 2006, pages 333-345) and US2003/181358 disclose the use of the haemoglobin of Arerzicola marina as a blood substitute. 45 [0010] FR2917292 discloses dressings comprising a haemoglobin of a vertebrate or an invertebrate. [0011] WO2010/122080 discloses a receptacle and a bioreactor comprising oxygen-carrying molecules. [0012] A model of quaternary structure of this extracellular haemoglobin fromArenicola marina has been proposed by Zal et al. (Zal et al. Eur. J. Biochem 1997, 243:85). The study indicated the existence of ten subunits: eight of which are globins, including two monomers (M=∼15 kDa) and five disulfide bonded trimers (T=~49 kDa). The remaining two 50 chains correspond to linker chains (L) that are disulfide bonded to form homo and hetero dimmers (~50 kDa). Three and six copies of each of the two monomers subunits and one copy of the trimer form a dodecamer subunit (D) with a mean mass close to 200 kDa. Twelve such complexes of globin chains are linked together by 42 linker chains to reach a total mass of 3648 6 24 kDa. [0013] While studying another family of polychaete annelids, the inventors isolated a novel haemoglobin from the 55 Nereididae family, that presents the advantage of being stable and functional for a longer period of time in several media. The worms belonging to the Nereididae family differ from those belonging to the Arenicolidae family for several aspects starting by their phylogeny. From a point of view of evolution, these two families have diverged from each other since several million years ago (Halanych and Janosik, 2006). Furthermore, these two annelid families do not have the same 2 EP 2 427 483 B1 morphology, the same behavior, the same genes, the same physiology neither the same organism biology. For example, Nereididae are errant species compared to the Arenicola worms which live in sand gallery on the intertidal area. Fur- thermore, Arenicolidae reproduce several times during their lifespan whereas Nereididae reproduces once the year and dies after the reproduction period. 5 [0014] The inventors then found that this novel isolated haemoglobin from the Nereididae family can be used as oxygen carrier for several applications, in particular for cell culture media complement, or as a supplement for organ preservation solution. SUMMARY OF THE INVENTION 10 [0015] One object of the invention is a cell culture medium comprising an isolated haemoglobin or a portion thereof from worms belonging to the family of Nereididae, having: - a P50 from 5 to 15, preferably from 9 to 12 mm Hg at 6-7°C, from 12 to 20, preferably from 15 to 18 mm Hg at 15 16-17°C and from 20 to 50, preferably from 30 to 44 mm Hg at 36-37°C - preferably no free cysteines, - preferably no glycosylations. [0016] In one embodiment of the invention, said haemoglobin is isolated from Nereis virens, also called Nereis diver- 20 sicola, Neanthes virens or Hediste diversicolor (although these scientific names are different they concern the same species) or other terms synonyms of these species. [0017] In another embodiment, said haemoglobin is in the form of an aqueous solution, a frozen solution, a thawed solution or a lyophilised powder. [0018] In one embodiment, said cell culture medium is a medium suitable for bioproduction. 25 [0019] In another embodiment, said cell culture medium is a medium suitable for cell growth.
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