Method for purification of natural cobalamins by adsorption on insoluble materials containing carboxylic groups

Field of invention

The present invention relates to a method for purification and/or concentration of natural cobalamins by adsorption on insoluble materials containing carboxylic groups.

Background of invention

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 α-site) is occupied by the 5',6'-dimethyl- benzimidazole base (Bzm). The upper axial position (β-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.

The industrial purification has until now been targeted to another form of the vitamin, CN-CbI, which contains cyano-group firmly bound to the β-site of CbI. The name "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.

The suggested method differs from the method described in the document WO

2006/024303, where another cobalt-coordinating group with different binding properties was used. In WO 2006/024303 H ₂ O-CbI is described to be purified from crude extract using a tetrazole containing matrix as Cbl-coordinating compound. However, it has turned out that 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. By using the present invention to purify cobalamins, it has surprisingly turned out that it is possible to obtain the cobalamins in a much easier and cheaper process compared to previously used methods, and also with essentially decreased or completely excluded application of the polluting and toxic cyanide.

Summary of invention

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

• providing a first solution comprising at least one type of natural cobalamins,

• providing an adsorbent material comprising carboxylic groups capable of making specific interaction with said natural cobalamins,

• contacting said first solution comprising at least one type of natural cobalamins with said adsorbent material, whereby at least a part of said at least one type of

natural cobalamins becomes adsorbed and/or bound to said adsorbent material, and

• elution of said at least one type of natural cobalamins from said adsorbent material, hereby • obtaining a concentrated solution comprising at least one cobalamin.

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.

Consequently, all natural cobalamins at a pH-value around 3.5 would bind to COOH- adsorbents essentially better than CN-CbI due to the additional RCOOH-cobalt

bonding. 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.

Description of Drawings

Fig. 1. Structure of CbI and its axial coordination positions. A) Structure of CbI, with Bzm at the α-position and an R-group at the β-position. B) H ₂ O-CbI. C) AdO-CbI and Me-CbL D) CN-CbI.

Fig. 2. Binding of CbI to CM-Sepharose at different pH. A) Binding at low ionic strength B) Binding at high ionic strength. C) Titration of CM-Sepharose suspension matrix:water = 1 :1 with NaOH.

Fig. 3. Binding of CbI to Amberlite Cobalamion. A) Removal of different cobalamins from solution at pH 3.5 B) Removal of H ₂ O-CbI from solution at different pH and ionic strength. C) Binding of CbI from fermentation broth with or without cyanide treatment on Cobalamion resin.

Fig. 4. Purification of CbI from fermentation broth. A) Scheme of the purification procedure. B) Absorbance spectra of the products obtained at different purification steps.

Detailed description of the invention

In a first aspect the present invention relates to a method for obtaining a concentrated solution comprising at least one cobalamin, the method comprising

• providing a first solution comprising at least one type of natural cobalamins, • providing an adsorbent material comprising carboxylic groups capable of making specific interaction with the natural cobalamins,

• contacting the first solution comprising at least one type of natural cobalamins with the adsorbent material, whereby at least a part of the at least one type of natural cobalamins becomes adsorbed and/or bound to the adsorbent material, and • elution of the at least one type of natural cobalamins and/or CN-CbI from the adsorbent material, hereby

• obtaining a concentrated solution comprising at least one cobalamin.

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.

In an embodiment 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).

In another embodiment 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 (wild type as well as recombinant) 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.

In a further embodiment 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). To minimize this competitive coordination, pH of solution should be low as described above (e.g. a pH 3 - 3.5) to protonate the above substances. No cyanide is present in the living cell, because this compound is extremely poisonous. It is possible to treat cell extracts with cyanide, and afterwards adsorb CN-CbI on an unspecific adsorbent material.

In an embodiment 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.

In a further embodiment 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.

In another embodiment 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.

In an embodiment 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. At the same time, 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 (β- and α- 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 β-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). In the development of the present invention it has been realised that all biological forms of CbI can be efficiently adsorbed on a

COOH-resin via bonding with cobalt. On the contrary, the cyanide treated vitamin (CN-CbI) is completely protected. Neither its upper cyano-group nor the Bzm-base can dissociate from cobalt under some of the non-destructive conditions as described elsewhere herein (Fig.1 D). Thus, depending on the parameters used in the method.the COOH-group of the absorbent material weakly binds CN-CbI via hydrogen bonding with amides of the corrin ring, but not cobalt ion. If using the low pH 2-4 CN-CbI weakly and unspecifically binds to the COOH-group.

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.

If 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.

In an embodiment 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.

As described above, 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.

The binding of Ado-Cbl and H ₂ O-CbI is under the mentioned conditions, i.e. with a low pH, approximately tenfold faster and twentyfold stronger than for CN-CbI. During a column adsorption from broth up to about 90% binding of the cobalamins of the first solution was observed after approximately 30 min, therefore 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.

In macroreticular resins very small gel beads may be crosslinked together and produce a porous net. In the present invention these 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.

In a further embodiment the adsorbent material may comprise an active monomer and a co-polymer. The active monomer may be selected from the group of, e.g. acrylic acid (CH ₂ =CH-COOH), methacrylic acid (CH ₂ =C(CH ₃ )-COOH) and vinyl benzoate (CH ₂ =CH(C ₆ H ₄ )-COOH) or a combination thereof.

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.

In an embodiment 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.

In an embodiment 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.

In a preferred embodiment the active monomers, acrylic acid, methacrylic acid, and/or vinyl benzoate, may be co-polymerized with one or more components 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 and/or with acrylonitryle, methylsterene, butadiene, styrene, divinylbenzene (DVB), divinylether (DVE)) to give a crosslinked resin.

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.

In an embodiment 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. The relative volume 1V corresponds to the volume occupied by beads (including the inter-beads space) in a column when using adsorption protocol with the ratio broth :adsorbent material = 40:1.

When bound to the adsorbent material the adsorbent material may be rinsed. 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. In a preferred embodiment 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.

In a further embodiment 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.

When eluting the bound cobalamins with an alkaline solution, this alkaline solution can be used in a volume of 2-50 bed volumes of the adsorbent material. Preferred is 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.

In respect of K ₃ PO ₄ 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. In respect of 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.

In an embodiment the alkaline solution may contain cyanide. The cyanide can be KCN and/or NaCN. Examples of 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.

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.

In a preferred embodiment 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.

In an embodiment 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%. In a preferred embodiment 30-70% of the cobalamins in the first solution is obtained in the concentrated solution. In another preferred embodiment this concentration is 40-60%. In a further preferred embodiment this concentration is 45-55%.

In another embodiment 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.

In a further embodiment 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

• Precipitation of impurities with methanol, and

• Evaporation or rotor-evaporation of methanol, dissolving in water and

• Adsorption/absorption of impurities on a mixed ion-exchanger.

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.

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. To provide a final concentration of CbI of 0.1 - 10 mM, preferably 3 mM, a volume of methanol used can be 4 relative volumes V.

In an embodiment 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.

In an embodiment 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. In another preferred embodiment cyanide is not used in the elution process either. In a more preferred embodiment cyanide is not used in the first solution and is not used in the elution process. In a most preferred embodiment cyanide is not used at all in respect of the processes described herein.

In an embodiment 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.

In another aspect the present invention relates to use of the method which is described herein.

In an embodiment the method as described herein can be used for purification and/or isolation and/or concentration at least one type of a cobalamin.

In a further aspect 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.

In a preferred embodiment the concentrated solution comprises less than 25% CN- cobalamin in respect of the total amount of cobalamins. In a more preferred embodiment the content of CN-cobalamin is less than 15%. In a further preferred embodiment the content of CN-cobalamin is less than 10%. In an even more preferred embodiment the content of CN-cobalamin is less than 5%. In the most preferred embodiment the content of CN-cobalamin is substantially 0.

In another aspect the present invention relates to the use of the concentrated solution as described elsewhere herein.

In a preferred embodiment 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.

Detailed description of the drawings

Fig. 1. Structure of CbI and its axial coordination positions. A) Structure of CbI, where the lower axial position (α) is occupied by Bzm nucleotide base, and the upper axial position (β) contains an active group R. All CbI forms can produce hydrogen bonds with protonated carboxylic groups R-COOH. B) Coordination pattern of H ₂ O-CbI. A nucleophilic ligand L substitutes for water at the β-site. C) Coordination patterns of

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 β-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. A) Binding at low ionic strength (10 imM buffers, phosphate, acetate, Tris, carbonate). Solutions of 200 μM H ₂ O-CbI or Ado-Cbl were incubated with CM-Sepharose at the ratio 10:1 for 3 min (22 ⁰ C). The samples were centrifuged, briefly washed with 1 mL H ₂ O, and the bound CbI was eluted in 0.2 M phosphate buffer, pH 12. Controls correspond to the binding pairs:

H ₂ O-Cbl+Sepharose 4B, CN-Cbl+CM-Sepharose. B) Binding at high ionic strength (50 mM buffers). Other conditions as in panel A. 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. A) Removal of different cobalamins from solution at pH 3.5, I = O M, 22 ⁰ C. Water samples of 400 μM H ₂ O-CbI, Ado-Cbl and CN-CbI were incubated with the resin at the volume ratio liquid:resin = 10:1 , and decrease of free CbI was monitored over time. Final ratios Cblf _re e/Cblbound corresponded to 0.02 (Ado-Cbl), 0.03 (H ₂ O-CbI) and 0.5 (CN-CbI). B) Removal of H ₂ O-CbI from solution at different pH and ionic strength (22 ⁰ C). Final ratios Cblfree/Cblbound corresponded to 0.03 (pH 5.5, I * 0 M), 0.04 (pH 3.5, I * 0 M) and 0.2 (pH 3.5, I * 0.1 M). All other conditions as in panel A. C) Binding of CbI from fermentation broth. The extract (pH 3.7, 22 ⁰ C, with or without cyanide treatment) was incubated in batch with

Cobalamion resin at the ratio liquid:resin = 10:1. The bound CbI was measured after 2 h and 5 h of adsorption, when the resin with bound CbI was treated with 0.5 M K ₃ PO ₄ , 5 imM KCN and absorbance of the eluted dicyano-Cbl was measured (ε ₅₇ 9= 9 900 M ^{" 1} cm ^"1 ). After 2 h of adsorption from broth two samples were subjected to washing with 3 x H ₂ O, 1 ^χ 20% 2-butanol, 2 x H ₂ O (in all cases the ratio liquid:resin = 10:1 ). The concentration of CbI was determined by measuring the absorbance spectrum of the washing liquids after overnight treatment with 0.1 M K ₃ PO ₄ , 5 mM KCN (ε ₅₇₉ = 9 900 M ^{" 1} cm- ¹ ).

Fig. 4. Purification of CbI from fermentation broth. A) Scheme of the purification procedure. The method as outlined is further described in Example 4. B) Absorbance spectra of the products obtained at different purification steps. Before measurements, the samples were diluted with water by factor 70 - 100. 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).

Examples

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.

Best results were obtained with Amberlite Cobalamion. We demonstrate that this resin has high affinity for the biological forms of CbI whereas the industrial form of the vitamin, i.e. CN-CbI, binds to Cobalamion much weaker. We have successfully purified CbI from a crude cell extract by the suggested method which was superior to the standard procedure.

Example 1. Test binding to CM-Sepharose.

The initial experiments indicated that the Amberlite resins containing carboxylic groups (R-COOH) could interact with cobalamins (e.g. CN-CbI) in an unspecific manner via side chains of the corrin ring, as explained in Introduction. Therefore, the specific axial binding of COOH-groups to the α/β-sites of CbI was tested with CM-Sepharose, a R-COOH containing matrix, where no side effects of interaction were observed. Thus, the control tests revealed that CM-Sepharose did not bind CN-CbI at all (bottom curves in Fig.2A, B). Likewise, the basic material Sepharose 4B (a matrix without carboxylic groups) did not interact with H ₂ O-CbI. This means, that the binding of natural cobalamins to Sepharose, if there was any, could be ascribed exclusively to their specific interaction with COOH-groups of the matrix.

CM-Sepharose adsorbed a noticeable amount of H ₂ O-CbI from solution at pH 3 - 8, see Fig.2A and B, upper curves. The cofactor form Ado-Cbl bound to CM-Sepharose at lower degree and only at pH<4 (Fig.2A). These data agree with the expected binding mechanisms shown in Fig.1 A and 1 B. Adsorption was noticeably better at low ionic strength (10 mM buffers, Fig.2A) when compared with high ionic strength (50 mM buffers, Fig.2B). Titration of COOH-groups in suspension of CM-Sepharose (matrix:water = 1 :1 ) is presented in Fig.2C. Total concentration of COOH-groups in the packed matrix was estimated as 100 mM. Multiple acid-basic equilibria with at least three pK were required to fit the titration curve: PK ₁ = 3.0 (30%), pK ₂ = 4.6 (48%) and pK ₃ = 6.0 (24%). It seems, that the most efficient interaction between CM-Sepharose and H ₂ O-Cbl/Ado-Cbl was achieved when both deprotoneted and protonated groups (R-COO ^" and R-COOH) were present in the matrix (Fig.2C). This observation may point to a more complex mechanisms of coordination, than those presented in Fig.1 B, C. For instance, several carboxylic residues may be involved in the binding of one CbI molecule, where a network of hydrogen bonds at the surface of CbI stabilizes the ligand-matrix contacts.

The preliminary data testified 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.

Three modifications of COOH-Amberlite (Cobalamion, IRC-76 and IRC-50) were used in the preliminary test, where the most suitable adsorbent was selected. The beads attached H ₂ O-CbI with the following rate coefficients 1.2 min ^"1 (Cobalamion), 0.3 min ^"1 (IRC-76) and 0.15 min ^"1 (IRC-50), when performing batch binding in water at the ratio liquid:resin = 4:1 , pH 3.5, 22 ⁰ C. The best results were obtained with Amberlite Cobalamion, therefore the following work was continued with this binding material.

Solutions of three cobalamins H ₂ O-CbI, Ado-Cbl and CN-CbI (400 μM) were incubated with Cobalamion under constant mixing at the ratio liquid:resin = 10:1 (water, pH 3.5, 22 ⁰ C). Decrease in the concentration of free CbIs was followed over time by measuring its absorbance (Fig.3A). The performed experiments demonstrated that H ₂ O-CbI and Ado-Cbl were removed from the medium faster and to higher degree than CN-CbI. This result agreed with the test performed previously with CM-Sepharose. Washing of the saturated resin with water also demonstrated difference in retention of Ado-Cbl and H ₂ O-CbI on one hand, and CN-CbI on another. Thus, a noticeable leakage of CN-CbI from the beads was observed during the washing procedure, whereas no significant liberation of two other ligands was detected. This means that the industrial adsorbent Amberlite Cobalamion binds the natural forms of CbI better than CN-CbI.

To evaluate the CbI binding at different pH, the beads were incubated overnight in 1 M phosphate buffer, pH 6.8, whereupon they were extensively washed with water. Finally, the resin was equilibrated with water and the measured pH was 5.5. Binding of H ₂ O-CbI to Cobalamion at pH 5.5 did not essentially differ from adsorption at pH 3.5 (Fig.3B). Similar velocities of two processes may indicate that the rate limiting step of attachment is diffusion of H ₂ O-CbI into the resin particles, but not the interaction of H ₂ O-CbI with the COOH-groups. Presence of salt (I ~ 0.05 M, pH 3.5) decreased efficiency of adsorption (Fig.3B).

Example 3. Binding of CbI from fermentation broth to Amberlite Cobalamion.

After the preliminary experiments with adsorption of pure cobalamins, we have carried out a comparative binding assay on fermentation broth provided by a manufacturer of B ₁₂ . The solution contained either natural cobalamins or CN-CbI prepared by treatment with cyanide. In both cases adsorption was conducted in batch at the ratio liquid:resin = 10:1 (pH 3.7, 22 ⁰ C). Incubation for 5 hours was sufficient to guarantee binding of approximately 95% and 60% of CbI without and with cyanide treatment, respectively, Fig.3C. Both binding processes were relatively slow, which can be explained by noticeable contamination of the resin with other components of the cell extract. Presence of contaminants on the surface of Cobalamion beads decelerated permeability of CbI through the pores of the beads and made this process rate limiting. Nevertheless, the natural cobalamins from broth bound to Cobalamion 10-15-fold better than CN-CbI according to the final level of adsorption, 95% and 60% respectively.

Washing of the Cbl-saturated resin with several portions of H ₂ O, 20% 2-butanol, H ₂ O removed the main amount of impurities, however some leakage of CbI from the adsorbent was also observed (Fig.3C). Loss of the ligand was quite insignificant in the case of natural cobalamins, whereas leakage of CN-CbI accounted for one third of the bound ligand.

All in all, retention of the natural cobalamins by Cobalamion was more efficient than that of CN-CbI. This allowed 1 ) capturing of the ligand at higher ratio liquid:resin and 2)

extensive washing of the resin without losses of CbI. Consequently, preparations of CbI with higher yield, concentration and purity could be obtained.

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 (including the inter- beads space) was assigned as one relative unit of volume (1V). Fermentation broth (pH 3.7, 22 ⁰ C, 40V, CbI = 300 μM) was applied to the Cobalamion column at the flow 2V / hour. 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. Optionally, 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 ^χ 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.

The choice of optimal eluent depends on the following purification strategy. For example, 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. In the absence of KCN, 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. In our purification procedure 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.

The Cbl-sample in 15% NH ₄ OH with 10 mM KCN was evaporated in a fume box at 65 ⁰ C under air flow. Methanol (100%, 4V) was added to dissolve CN-CbI, and an insoluble brown contaminant was precipitated by centrifugation. Methanol was evaporated in a fume box at 65 ⁰ C under air flow, and the dry rest was dissolved in water. The absorbance spectrum confirmed removal of the brown contaminant, according to better correspondence of the experimental sample with the standard CN CbI at 450 - 550 nm (Fig.4B, curve 7). At the same time, the UV record at 250 - 350 nm still demonstrated presence of contaminating compounds. These impurities were removed by ion-exchange chromatography conducted with a mixed Amberlite resin (1V), composed of three components taken in equal volumes: strong anion exchanger 900 ( reticular form, Cl ^" ), strong cation exchanger 200 ( reticular form, Na ⁺ ) and strong cation exchanger component of IRN-150L (gel form, Na ⁺ ). Analogous products with other trademarks can be used as well. Chromatography was conducted at the flow 1 V / hour. Before and after application the resin was washed with 2V of 5 M NaCI. The filtered solution was depleted of UV-contaminants and the obtained preparation of CN-CbI did not differ from the standard CN-CbI (Fig.4B, curve 8 and the standard). The final preparation of CbI corresponded to 70% of the original cobalamins in the fermentation broth.

The shown model purification demonstrates that application of the described adsorption method is feasible for isolation of CbI from crude mixtures.