Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/265—Adsorption chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
  • B01J20/26—Synthetic macromolecular compounds
  • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
  • B01J20/282—Porous sorbents
  • B01J20/285—Porous sorbents based on polymers
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12

Definitions

• the present invention relates to a method for purification and/or concentration of natural cobalamins by adsorption on insoluble materials containing carboxylic groups.
• Vitamin B 12 (or cobalamin, CbI) is an organometallic cofactor of complex structure. It is synthesised only by bacteria, and all other organisms obtain the vitamin via a complicated food chain. Insufficiency of B 12 in humans causes severe disorders accompanied by neurological abnormalities, anaemia and final death.
• the core structure of CbI (Fig.1A) includes the corrin ring with the central cobalt ion. Its lower coordination positions (called also ⁇ -site) is occupied by the 5',6'-dimethyl- benzimidazole base (Bzm). The upper axial position ( ⁇ -site), may contain different groups bound to cobalt with varying strength. Yet, all variants of CbI are converted inside the animal cell to the cofactors methyl- and 5'-deoxy-5'-adenosyl-cobalamin (Me-CbI, Ado-Cbl, respectively). The two above forms of cobalamin undergo under physiological conditions an occasional transformation to H 2 O-CbI, vitamin B 12a . Therefore, Ado-Cbl, Me-CbI and H 2 O-CbI are three ubiquitous variants of the natural cobalamins.
• vitamin B12 belongs, strictly speaking, to CN-CbI.
• This form is prepared by the treatment of bacterial extracts with KCN or NaCN and conversion of the biological cobalamins to CN-CbI.
• Vitamin B12 is produced industrially by microbial fermentation, using almost exclusively recombinant Pseudomonas denitrificans and Propionibacterium species, then converting the natural cobalamins into the cyanocobalamin form by chemical processes including cyanidation followed by extraction and purification steps using organic solvents. The chemical conversion step and any subsequent purification steps cause this production process to be expensive, unsafe to the operators and environmentally unfriendly.
• H 2 O-CbI is described to be purified from crude extract using a tetrazole containing matrix as Cbl-coordinating compound.
• the material containing tetrazole does not bind Ado-Cbl and Me-CbI, and cell extract has to be illuminated intensively to convert other cobalamins to H 2 O-CbI. Additional drawback originates from a relatively high price of the tetrazole-containing material.
• the present invention describes a method for purification of cobalamins, where the method has optimal conditions of interaction between natural cobalamins and a material containing COOH-groups.
• the material may be a commercially available material such as Amberlite resins containing COOH-groups.
• the invention as described herein relates to use of a method and to the method itself, where the method is a method for obtaining a concentrated solution comprising at least one cobalamin, and where the method comprises the steps of
• the natural cobalamins which may be obtained can be any of 5'-deoxy-5'-adenosyl- Cobalamin (adenosyl-Cbl, Ado-Cbl), methyl-cobalamin (methyl-Cbl, Me-CbI) and aquo- cobalamin (aquo-Cbl, H 2 O-CbI).
• the cobalamins can be obtained using no or decreased amounts of cyanide when compared to other methods.
• a concentrated solution is obtained by the method described, this solution comprising at least one natural cobalamin and/or CN-CbI, which can be used for the preparation of a medicament, a dietary supplement and/or a vitamin preparation.
• Cobalamin (CbI, vitamin B 12 ) is a complex organic molecule necessary for human metabolism. Its industrial purification from the cyanide-treated bacterial extract is based on unspecific biding of CN-CbI to hydrophilic or hydrophobic adsorbents.
• the presented invention allows to increase the specificity of interaction between CbI and the COOH-containing adsorbents, e.g. Amberlite Cobalamion, which makes the purification procedure more efficient.
• the method is based on the ability of certain CbIs to bind to COOH-containing resins via a specific coordination bond between cobalt ion of CbI and COOH-group. This particularly concerns, the natural cobalamins, i.e.
• Adenosyl-Cbl (Ado-Cbl), Methyl-Cbl (Me-CbI) and Aquo-Cbl (H 2 O-CbI), which have turned out to bind to COOH-resins essentially better than CN-CbI.
• the water molecule of H 2 O-CbI is easily displaced from cobalt by COOH-groups at pH of e.g. 3 - 6.
• Two other biological forms (Ado- and Me-CbI) have a partially protected cobalt ion, however it has been found that they becomes accessible for coordination at pH ⁇ 4.
• the present invention is developed to improve adsorption of CbI from fermentation broth, decrease the amount of adsorbent, and to exclude the hazardous cyanide treatment of fermentation broth, because CN-CbI is incapable of the RCOOH-cobalt bonding.
• Fig. 1 Structure of CbI and its axial coordination positions.
• Fig. 2 Binding of CbI to CM-Sepharose at different pH.
• C) Titration of CM-Sepharose suspension matrix:water 1 :1 with NaOH.
• Fig. 3 Binding of CbI to Amberlite Cobalamion.
• Fig. 4 Purification of CbI from fermentation broth.
• the present invention relates to a method for obtaining a concentrated solution comprising at least one cobalamin, the method comprising
• the method as described herein can also be used to purify and/or isolate and/or concentrate natural cobalamins.
• the concentrated solution comprising at least one cobalamin may have impurities and the concentrated solution may be further treated e.g. as described herein below.
• the natural cobalamins can be one or more of the natural cobalamins 5'-deoxy-5'-adenosyl-Cobalamin (adenosyl-Cbl, Ado-Cbl), methyl- cobalamin (methyl-Cbl, Me-CbI) and aquo-cobalamin (aquo-Cbl, H 2 O-CbI).
• these cobalamins are by far the major forms of CbI present in the living cell with the following approximate distribution: 60% Ado-Cbl, 20% Me-CbI, 20% H 2 O-CbI.
• the cobalamins obtained in the concentrated solution may comprise the main part as the original mixture of cobalamins (alkaline elution in the darkness), CN-CbI (alkaline elution in the presence of KCN or NaCN), or H 2 O-CbI (alkaline elution with following illumination).
• the first solution comprising at least one type of natural cobalamins may be a solution selected from the group of fermentation broth; cell extract e.g. a culture supernatant or a filtrate; biological extract; a liquid of arbitrary composition containing natural cobalamins either pure or with contaminating compounds; or a mixture thereof.
• the mentioned types of solutions based on microorganisms may be based on bacterial cultures and/or recombinant yeasts.
• the bacteria Pseudomonas denitrificans and
• Propionibacterium species can be used to produce a fermentation broth.
• a biological extract can also be a plant extract from recombinant plants and/or an extract from animal or human tissues (e.g. liver, kidney, muscle) or animal or human liquids (e.g. blood plasma, milk, saliva).
• An extract or liquid from an animal may be obtained from the mentioned organs of any animal preferred is from pigs and cows.
• the first solution as described herein may be pre-treated before contacted with the adsorbent material.
• a pre-treatment may be a filtration to remove debris, e.g. components from cell walls, cell membranes and/or cell organelles.
• the first solution comprising at least one type of natural cobalamins may have a pH-value of between about 2 and about 8 when contacting the first solution with the adsorbent material.
• Preferred is a pH-value of between 3 and 6.
• Preferred is also a pH-value of between about 2.5 and about 4.5. More preferred is a pH-value of between about 3 and about 4.
• Most preferred is a pH-value of about 3.5.
• Destruction of cells and homogenization exposes H 2 O-CbI to different nucleophiles capable of coordination, e.g. histidine, histidine- and/or cysteine-containing peptides, and reduced glutathione (GSH).
• pH of solution should be low as described above (e.g.
• the first solution comprising at least one type of natural cobalamins has an ionic strength of about 0 to about 0.5 M when contacting the first solution with the adsorbent material.
• Preferred is an ionic strength of about 0 to about 0.25 M. More preferred is an ionic strength of about 0 to about 0.1 M.
• the first solution comprising at least one type of natural cobalamins has a temperature of 5 - 5O 0 C when contacting the first solution with the adsorbent material.
• the temperature may also be higher e.g. up to 90 0 C or 100 0 C.
• An increased temperature accelerates the binding process.
• Preferred is a temperature of between 10 and 4O 0 C. More preferred is a temperature of between 15 and 35 0 C. Even more preferred is a temperature of between 20 and 3O 0 C. Most preferred is a temperature of about 25 0 C or room temperature.
• the first solution comprising at least one type of natural cobalamins further comprises an organic solvent.
• This organic solvent may be an organic alcohol or acetone. Among organic solvents the following may be used alone or in combination: methanol, ethanol, propanol, butanol.
• the amount of the organic solvent may be 1-50%, such as 5-30%, e.g. 10-20% of the volume of the first solution.
• At least one type of natural cobalamins becomes adsorbed and/or bound to the adsorbent material due to specific adsorption and/or binding between a cobalt atom of the natural cobalamins and the carboxylic groups of the adsorbent material and/or due to weak hydrogen bonding.
• All cobalamins can potentially interact with polymers, e.g. via weak hydrogen bonding between the amide side chains of the corrin ring and the protonated carboxylic groups (R-COOH) of an adsorbent, as shown in Fig.1A.
• R-COOH protonated carboxylic groups
• this type of binding is weak, unspecific and requires the correct size of pores inside the resin.
• a stronger binding can be achieved between certain forms of CbI and COOH-resins.
• This type of interaction involves the upper and/or lower axial positions of CbI ( ⁇ - and ⁇ - sites, respectively), which can be either open or closed for coordination, depending on the form of CbI and the binding conditions.
• H 2 O-CbI contains a ⁇ -coordinated water molecule, which is weakly associated with cobalt and can be readily displaced by other chemicals (Fig.1 B). It has turned out that H 2 O-CbI can potentially bind via its upper surface to adsorbents with nucleophilic groups, for instance a COOH-residue. The two catalytic forms (Me-CbI and Ado-Cbl) are protected from coordination at the upper side. Yet, their Bzm base can dissociate from the lower surface of the corrin ring at acidic pH of about 2 - 4 which makes the cobalt ion open for coordination (Fig.1 C).
• Cobalt-coordinating properties of the biological cobalamins can be used for their purification from crude cell extracts. This method would be essentially advantageous in comparison with weak and unspecific binding of CN-CbI to hydrophilic or hydrophobic resins. Thus conversion to CN-CbI essentially weakens interaction of cobalamin with the carboxylic groups containing adsorbent material as described herein.
• Cyano-cobalamin (CN-CbI) is present in the first solution, CN-CbI may become bound to the adsorbent material by weak hydrogen bonds in an unspecific manner. Natural cobalamins can bind to the adsorbent material due to specific interaction between the cobalt ion of the cobalamin and COOH-residues of the adsorbent material. Also hydrogen bonding may be involved in the binding of the natural cobalamins to the adsorbent material. The different binding systems of cyano-cobalamin and the natural cobalamins make a difference for the achieved binding velocity and affinity, which may be essentially higher for the natural cobalamins.
• the adsorbent material comprising carboxylic groups can be a polymeric material including free carboxylic groups.
• Preferred is a concentration of carboxy-groups of 0.5-5 mol/L of wet material. More preferred is a concentration of 0.7- 4 mol/L of wet material. Most preferred is a concentration of 1-3 mol/L of wet material.
• the pH of the first solution may be of between 2 and 8, whereas inside and/or close to the adsorbent material the pH is expected to have a pH of about 2 to about 3 substantially irrespectively to the pH of the first solution.
• the volume inside and/or close to the adsorbent material may be buffered by the COOH-groups of the adsorbent material.
• Ado-Cbl and H 2 O-CbI are under the mentioned conditions, i.e. with a low pH, approximately tenfold faster and twentyfold stronger than for CN-CbI.
• a flow of the first solution can be 1 - 2 bed volumes per hour.
• the polymeric material as described herein may be in any form, e.g. a reticular form and/or in the form of beads.
• pores may have a diameter suitable for the cobalamin molecules to penetrate the material.
• the pores may have a diameter of e.g. 100-2000 A, such as 150-1500 A, e.g. 200-1000 A, such as 250-900 A, e.g. 300-800 A, such as 350-700 A, e.g. 400-600 A, such as 400-500 A.
• Small beads made of a material as described herein can produce larger beads e.g. with a diameter of 0.1 to 1 mm.
• Beads size can be measured in "mesh".
• Mesh means number of units per inch. For instance, 50 mesh would correspond to a particle of ⁇ 0.5 mm.
• the larger particles of the present invention may have a diameter of 0.1-3 mm. Preferred is a diameter of 0.2-2 mm. More preferred is a diameter of 0.3-1 mm. Most preferred is a diameter of about 0.5 mm.
• the adsorbent material may comprise an active monomer and a co-polymer.
• the acryl components used as polymeric material may be selected from the group of acrylic acid, butyl acrylate, 2-ethylhexyl acrylate, methyl acrylate, ethyl acrylate acrylonitrile, methyl methacrylate and/or tri-methylol-propane-triacrylaet or a combination thereof.
• the polymeric material may further comprise styrene, vinyl, acryl, and/or saccharide components. Also the polymeric material may comprise acrylonitryle, methylsterene, butadiene, styrene, divinylbenzene (DVB), and/or divinylether (DVE) to give a crosslinked resin.
• styrene vinyl, acryl, and/or saccharide components.
• polymeric material may comprise acrylonitryle, methylsterene, butadiene, styrene, divinylbenzene (DVB), and/or divinylether (DVE) to give a crosslinked resin.
• the active monomer as described above comprises 1-90% of the absorbent material.
• the amount of active monomer may be 1-80% of the total amount of polymeric material, such as 1-70%, e.g. 1-60%, such as 1-50%, e.g. 1-40%, such as 1 -30%, e.g. 1 -20%, such as 1 -10%. Also the amount of the active monomer may be e.g. 5-10%, 10-15%, 15-20%.
• Substantially all the part of the absorbent material not being an active monomer may be a co-polymer. Any suitable co-polymer may be used e.g. at least one acryl component. Acryl components may be the one as described elsewhere herein.
• the active monomers acrylic acid, methacrylic acid, and/or vinyl benzoate
• acrylic acid butyl acrylate
• 2-ethylhexyl acrylate methyl acrylate
• ethyl acrylate acrylonitrile methyl methacrylate and/or tri-methylol-propane-triacrylaet and/or with acrylonitryle
• methylsterene butadiene
• the polymeric material described herein may be a commercially available product which has and/or can be modified to obtain a large numbers of carboxylic groups.
• Examples of commercial products are Amberlites products such as Amberlites Cobalamion, IRC-50, IRC-76, and/or CM-Sepharose.
• the adsorbent material as described herein may prior to use be pretreated. Pretreatment may be a regeneration of the resin. In a preferred embodiment the regeneration is performed with an acidic solution. In a more preferred embodiment the regeneration is performed with HCI. The regeneration may be performed with 1 M of HCI.
• the volume of the acidic solution may be any suitable, e.g. 1-5 bed volume of the solution. In a preferred embodiment HCI of 2 bed volume can be used.
• the binding of the at least one type of natural cobalamins to the adsorbent material is conducted by means of column chromatography and/or adsorption in batch and/or membrane filtration.
• Ratio broth : adsorbent material may be from 200:1 to 1 :1 , preferably 40:1.
• a rinsing or washing solution may be water, an alcohol, and/or acetone. Rinsing solutions of water may use water of 20°C or it may be heated e.g. to between 25°C and 70°C.
• An alcohol may be ethanol, propanol (1- or 2-), butanol (1- or 2-), and/or tert-butanol. One or more rinsing solutions may be used separately or mixed.
• the adsorbent material is rinsed with water followed by alcohol and again with water.
• the concentration of the alcohol may be 10-100%, preferentially 15-25% or substantially 20 %.
• An example of a rinsing solution is 20% of 2-butanol.
• the volume of the rinsing solution may be any suitable to rinse the adsorbent material.
• the volume may be 2-50 bed volumes in total, such as 3-40 bed volumes, e.g. 4-30 bed volumes, such as 5-20 bed volumes, e.g. 6-10 bed volumes.
• At least one type of natural cobalamins when bound to the adsorbent material are eluted from the adsorbent material with an alkaline solution.
• the alkaline solution may be any suitable solution and may be selected from the group of NH 4 OH, K 3 PO 4 , NaOH, KOH, Tris, triethylamine, bicarbonate, an amine-containing compound, or a combination thereof.
• the elution may also be performed by a stepwise application of alkaline solutions e.g. first a volume of NH 4 OH then a volume of KOH.
• Such stepwise use of alkaline solutions may be in any order with the alkaline solutions mentioned herein.
• this alkaline solution can be used in a volume of 2-50 bed volumes of the adsorbent material.
• a volume of 2-40 bed volumes More preferred is a volume of 2-30 bed volumes. Even more preferred is a volume of 2-20 bed volumes. Most preferred is a volume of 2-10 bed volumes.
• the alkaline solution can have a high molarity of between 0.2 to 10 M. Preferred is a molarity of 0.3-9 M. More preferred is a molarity of 0.4-8 M. Even more preferred is a molarity of 0.5-7 M. Further preferred is a molarity of 0.6-6 M. Preferred is also a molarity of 0.7-5 M. More preferred is a molarity of 0.8-4 M. Even more preferred is a molarity of 0.9-3 M. Further preferred is a molarity of 1-2 M.
• a preferred molarity of the alkaline solution is 0.2-5 M.
• Preferred is a molarity of 0.5-4 M. More preferred is a molarity of 1-3 M. Even more preferred is a molarity of about 2 M.
• NH 4 OH a preferred molarity of the alkaline solution is 1-10 M corresponding to 3-30%.
• Preferred is a molarity of 2-9 M. More preferred is a molarity of 3-8 M. Even more preferred is a molarity of about 4-7 M. Further preferred is a molarity of about 5-6 M.
• the alkaline solution may contain cyanide.
• the cyanide can be KCN and/or NaCN.
• concentration of cyanide can be 1 - 200 mM concentration, also the concentration may be of 1-25 mM, 25-50 mM, 50-75 mM, 75-100 mM, 100-150 mM or 150-200 mM, preferably 5-10 mM.
• cyanide If cyanide is used, this binds to cobalt ion of Cobalamin. Binding of cyanide completely precludes specific coordination of R-COOH to cobalt ion of cobalamins. It may also be possible to avoid cyanide in the elution medium. In this case the eluted product would be a mixture of H 2 O-CbI, Ado-Cbl and Me-CbI, where H 2 O-CbI is expected to be the prevailing form, if no protection from light is provided. In this way, purification can be targeted to H 2 O-CbI.
• the alkaline solution may be selected from 3-30% NH 4 OH (1 -10 M) with or without 1 -1 OO imM cyanide, or 0.2-5 M K 3 PO 4 with or without 1 -100 imM cyanide.
• the concentrated solution comprising at least one cobalamin may comprise at least 30% of the cobalamin comprised in the first solution.
• the concentration of the cobalamins in the concentrated solution may also be at least 40%, such as at least 50%, e.g. at least 60%, such as at least 70%, e.g. at least 80%, such as at least 90%, e.g. at least 95%.
• 30-70% of the cobalamins in the first solution is obtained in the concentrated solution.
• this concentration is 40-60%.
• this concentration is 45-55%.
• the concentrated solution comprising at least one cobalamin may have a concentration of at least 0.5 mM in respect of the at least one cobalamin.
• This concentration may also be at least 0.75 mM, such as at least 1 mM, e.g. at least 1.25 mM, such as at least 1.5 mM, e.g. at least 2 mM, such as at least 2.25 mM, e.g. at least 2.5 mM, such as at least 2.75 mM, e.g. at least 3 mM, such as at least 3.5 mM, e.g. at least 4 mM, such as at least 4.5 mM, e.g. at least 5 mM.
• the concentrated solution may be purified.
• Any suitable purification process may be used.
• An example of such a purification process comprises the steps of: • Evaporation or lyophilization of liquids and/or desalting, and
• the evaporation step may be performed by any known evaporation techniques and may include air flow or rotor evaporation under vacuum. Also heating the concentrated solution to increase the efficiency of evaporation can be performed. An evaporation may be performed in 3-12 hours. Desalting may include adsorption on charcoal or XAD-resins followed by elution with alcohol (e.g. 96% ethanol) and evaporation.
• alcohol e.g. 96% ethanol
• Removal of impurities may be obtained by dissolving the dried concentrated sample from previous step in 3-5 relative volumes V of methanol (80-100%, preferably 100%).
• the cobalamins are dissolved in methanol whereas impurities or contaminants remain in pellet and can be removed by centrifugation and/or filtration.
• a volume of methanol used can be 4 relative volumes V.
• rotor evaporation cam be performed at a temperature of 10-80 0 C, preferably 20 - 40 0 C, and under vacuum.
• the relative volume of the mixed ion exchanger corresponds to 1 V at approximately equal parts of three components: 1 ) 0.333 V of strong cation exchanger (reticular form) Amberlite 200C or analogous products, 2) 0.333 V strong anion exchanger (reticular form) Amberlite IRA-900 or analogous products, 3) 0.333 V strong cation exchanger component from Amberlite IRN-150L, alternatively 0.333 V strong cation exchanger (gel form) Amberlyst 131 or analogous products.
• the first solution which comprises at least one type of natural cobalamins is not treated with cyanide before contacting the first solution comprising at least one type of natural cobalamins with the adsorbent material.
• Cyanide is a toxic compound, thus in a preferred embodiment cyanide is not added to the first solution.
• cyanide is not used in the elution process either.
• cyanide is not used in the first solution and is not used in the elution process.
• cyanide is not used at all in respect of the processes described herein.
• a concentrated solution is used as the first solution, hereby subjecting the concentrated solution to the method again, and performing another concentration of the solution.
• the present invention relates to use of the method which is described herein.
• the method as described herein can be used for purification and/or isolation and/or concentration at least one type of a cobalamin.
• the present invention relates to a concentrated solution comprising at least one natural cobalamin, wherein the concentrated solution is obtained by the method described elsewhere herein.
• the concentrated solution comprises less than 25% CN- cobalamin in respect of the total amount of cobalamins.
• the content of CN-cobalamin is less than 15%.
• the content of CN-cobalamin is less than 10%.
• the content of CN-cobalamin is less than 5%.
• the content of CN-cobalamin is substantially 0.
• the present invention relates to the use of the concentrated solution as described elsewhere herein.
• the concentrated solution is used for the preparation of a medicament, a dietary supplement and/or a vitamin preparation.
• the concentrated solution may be pre-treated before incorporated into medicament, a dietary supplement and/or a vitamin preparation.
• a pre-treatment may comprise a purification.
• Fig. 1 Structure of CbI and its axial coordination positions.
• Ado-Cbl and Me-CbI Strong donation of electrons from Ado- or Me-groups to cobalt ion destabilizes coordination of Bzm and it can dissociate at low pH. Afterwards, certain ligands can coordinate to the lower ⁇ -position. D) Protection of the axial positions in CN-CbI precludes any coordination of the external ligands.
• Fig. 2 Binding of CbI to CM-Sepharose at different pH.
• C) Titration of CM-Sepharose suspension matrix:water 1 :1 with NaOH. Arrow indicates best conditions for H 2 O-CbI binding.
• Fig. 3 Binding of CbI to Amberlite Cobalamion.
• Fig. 4 Purification of CbI from fermentation broth.
• the absorbance spectrum of the eluted CN-CbI (fraction 3) indicates presence of contaminating compounds which absorb light in both UV (250-350 nm) and visible diapason of light (>350 nm).
• the contaminants characterized by absorbance in visible diapason are generally removed in fraction 7.
• the contaminants characterized by absorbance in UV-light are removed in fraction 8.
• the spectrum of the final fraction 8 is identical to that of commercially available preparation of CN CbI (Sigma).
• the method as described herein has been tested on raw fermentation broth, prepared by a manufacturer of B 12 according to our recommendations.
• the biological cobalamins were efficiently adsorbed at pH 3.7 on different COOH-Amberlites and eluted at pH 10 - 12 in the presence or absence of KCN.
• the obtained product had higher concentration and purity already after the first isolation step.
• Adsorption was noticeably better at low ionic strength (10 mM buffers, Fig.2A) when compared with high ionic strength (50 mM buffers, Fig.2B).
• the preliminary data test ified that the binding of biological cobalamins to CM- Sepharose was much more efficient than adsorption of CN-CbI (binding of the latter was actually absent). Therefore, the binding assay was extended from the soft Sepharose matrix to the rigid resins suitable for industrial application.
• Example 2 Binding of CbI to different Amberlite resins containing COOH-groups.
• Amberlite is a robust adsorbent made of beads capable to resist high pressure and aggressive media.
• the volume of adsorbent in our experiments was measured according to the space occupied by wet acidic beads in a measuring glass. The volume between the beads was included into this value.
• the active elements in the tested materials were carboxylic groups R-COOH, which gave pH 3 - 4 after equilibration of the beads with water.
• the concentration of active groups corresponded to 2 - 3 mol per 1 L of wet resin.
• Example 4 Purification of CbI from fermentation broth.
• a model purification of CbI from fermentation broth was conducted (Fig.4A).
• the first purification step was column adsorption of the natural cobalamins on Amberlite Cobalamion resin.
• the space occupied by beads inside the column was assigned as one relative unit of volume (1V).
• Approximately 90% of CbI was bound to the resin at the end of this procedure.
• the adsorbed material was sequentially washed with water (4V), 20% 2- butanol (4V) and water (4V), room temperature.
• washing with warm water (5O 0 C, 10V) is possible as an alternative, but removal of contaminants is less efficient.
• Elution of adsorbed CbI can be conducted in 3 - 4 steps using alkaline solutions of high molarity, e.g. 1 ) 15% NH 4 OH with or without 10 mM KCN (4 ⁇ 1V, 1 hour each step) or 2) 0.5 M K 3 PO 4 with or without 10 mM KCN (3x1 V, 8 hours each step). After elution, the resin was regenerated by washing with 2V of 1 M HCI.
• elution with K 3 PO 4 requires following desalting and is therefore less convenient.
• Presence of cyanide in the eluent causes conversion of natural cobalamins to their cyano- and dicyano-forms, which are the typical products of the industrial purification.
• the major form of CbI in the eluent is H 2 O-CbI (if no protection from light is used).
• the sample protected from light contains the original mixture of natural cobalamins.
• the solution of 15% NH 4 OH with 10 mM KCN was used.
• the eluted cobalamin was obtained in its dicyano-form because of excess of cyanide.
• Dicyano-Cbl underwent conversion to CN-CbI under dilution with water, neutral buffer, or after acidification of the medium.
• the major degree of purification was achieved already after the first step, according to the absorbance spectrum of the diluted product CN-CbI presented in Fig.4B (top curve 3).
• the spectrum indicates presence of contaminants, which absorbed light in UV-diapason (250 - 350 nm) and at visible wavelengths (>350 nm, brown substance).
• Absorbance record of the standard preparation of CN-CbI is shown for a comparison (Fig.4B, bottom curve).
• the detected impurities were removed according to the procedure described below, though, other approaches are also possible.

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