Methods to purify molecules containing corrin

Abstract

METHODS FOR PURIFYING MOLECULES CONTAINING CORRINA. The invention relates to affinity adsorbent materials that contain high specificity functional groups, in particular tetrazole, capable of binding to compounds containing corrin, such as cobalamin (Cbl, or vitamin B ~ 12 ~). The invention also relates to methods for making such adsorbent materials and their use in the purification of molecules containing corrin, such as cobalamin. Compositions containing cobalamin purified by the method are also provided.

Description

"METHODS FOR PURIFYING MOLECULES CONTAINING CORRIN" All patent and non-patent references cited in this application are incorporated herein by reference in their entirety. FIELD OF THE INVENTION The present invention relates to affinity adsorbent materials containing highly specific functional groups capable of binding corrin-containing compounds, such as cobalamin (Cbl, or vitamin B). 12 The invention also relates to methods for manufacturing such absorbent materials, and their use in the purification of corrin-containing molecules, such as cobalamin. Compositions containing cobalamin purified by the method are also provided. BACKGROUND OF THE INVENTION Vitamin B i2(or cobalamin, Cbl) is a biological cofactor involved in two important processes in mammals: restoration of methionine from homocysteine ​​and conversion of propionic acid to succinate (1). All mammals depend on external sources of Bi2 since it is synthesized only by microorganisms (2). Grass-eating animals obtain Cbl from numerous bacteria that inhabit their intestinal system. Other animals, including humans, obtain the vitamin from animal-based food sources such as meat, milk, and eggs (3, 4). A lack of B i2 causes severe disorders including neurological abnormalities and anemia (4, 5). Cbl is the most complex molecule among the cofactors (figure 1) and consists of three fractions: 1) the corrinoid ring with the cobalt atom tral, 2) the nucleotide loop coordinated through its base to cobalt in the lower axial position (α site), 3) 0 coordinated group for cobalt in the upper axial position (site 0). Microorganisms that produce B 12 They synthesize both cobalamin and its analogues, which differ in their structure from B. 12 and have no catalytic function in mammalian cells (2, 6). The most typical analogues are corrinoids with an incorrect nucleotide base (e.g., pseudo B 12 , factor A) or missing parts of the molecule (e.g., cobinamide). Several intermediates of cobalamin synthesis belong to this latter group. All cobalamins and most analogues are colored substances with characteristic absorbance spectrum, which simplifies the measurement of their concentrations and allows the different corrins (6) to be differentiated from each other. Biologically active Cbl with the correct backbone can contain different axial top groups coordinated to a cobalt atom at the (3) position (Figure 1). The most common (3) groups are: methyl, deoxyadenosyl, cyanide, water, as well as numerous other organic and inorganic compounds (6). All forms of Cbl with the correct backbone (regardless of the nature of their £ group) can be converted into the catalytically active cofactors methyl- or deoxyadenosyl-cobalamin (Cbl-Met, Cbl-Ado) by enzymes in interior of the cell (3). An intermediate form of Cbl du During this conversion, it is aquo-Cbl (Cbl-OH2, vitamin B 12a • Cbl-OH2, Cbl-Ado and Cbl-Met are therefore abundant cobalamins from any natural source (3, 4, 7). The cyano form of Cbl (Cbl-CN) is the typical product of industrial purification and is also called vitamin B i2 . The manufacture of Cbl is a well-established process, which includes numerous stages, leading to the isolation of the active substance from a bacterial suspension (8, 9). The exact details are not publicly available, but the main steps are considered to be: 1) fermentation; 2) thermolysis of the cells with added cyanide; 3) non-specific adsorption of small organic molecules (including Cbl) onto a matrix, for example, charcoal; 4) elution and adsorption onto another resin, for example, hydrophobic adsorbent XAD; 5) elution and extraction of Cbl from the aqueous phase to phenol; 6) extraction of Cbl from phenol by water-acetone mixture; 7) drying; 8) dissolution in hot water and crystallization of the acetone-water solution at room temperature; 9) final drying. The purified product is Cbl-CN (vitamin B 12 ), whereas preparation of Cbl-OH2 (vitamin B 12a ) requires additional chemical treatment and re-purification. The above-described procedure employs numerous non-specific steps for isolation. Instead, it could potentially exploit a specific characteristic of C-bl, namely, the ability to coordinate certain compounds at the (3) site. For example, a matrix with a specific [3] group attached could potentially be used to extract Cbl from a solution. This would mean that one could purify the Vitamin D from a crude extract in only one step. For successful application of this method, it is desirable that the Cbl in the source medium have a weakly associated upper axial ligand, which can be displaced by the associated adsorbent group. In the above-described form, the main forms of Cbl available from biological sources are Cbl-Met, Cbl-Ado, and Cbl-OH2. However, the carbon-cobalt bond of Cbl-Met and Cbl-Ado is sensitive to light even under diffuse illumination (6). As a result, both forms undergo spontaneous conversion in aqueous solution to Cbl-OH2 if no specific precautions are taken. The Cbl-OH2 obtained has in its P site the weakly associated molecule (H2O) which can be easily displaced by numerous chemicals (6, 10). Several known compounds can bind to Cbl-OH2 with different affinities, causing characteristic changes in the absorbance spectrum of Cbl. This simplified the determination of the dissociation constants of the complexes produced (6, 10): CN- (Kd < IO' 12 M), SO3 -2 (Kd = 10' 7 M), SH cis-teine ​​group (Ka = 10' 6 M) , NO2" (Kd = IO' 5 M), N3' (Kd = 10' 5 M), imidazole and histidine (Kd = 10'4 M), different amino groups (Kd = 10' 3 M) , and acetate (Kd = 1 M) . However, these simple inorganic ions probably lose the properties of binding Cbl after coupling to a matrix. Furthermore, some of them (SH-containing chemicals) initiate reduction and destruction of Cbl (6). An amino-sepharose resin was previously used to link Cbl-OH2 with low affinity (Nexo 1975, Biochim Biophys Acta 379:189-192). However, binding only occurred at high Cbl concentrations and was very slow (16-20 hours). This method, therefore, cannot be used to purify the vitamin from biological sources where the vitamin concentration is low. In conclusion, there is a need for a simple and rapid method to isolate Cbl and other corrin-containing compounds from biological and other sources. SUMMARY OF THE INVENTION In one aspect, the invention provides a suitable adsorbent material for the purification of a corrin-containing compound, wherein said adsorbent material: comprises a tetrazoline in the form of a functional group, and / or - It is obtainable by a method that includes the following steps: i) provide an activated matrix comprising cyanide, and ii) treat said matrix with a solution containing azide, and / or - has a specific functional group (3), wherein said functional group binds Cbl-OH2 at pH 3 to 7.5, at 5 °C to 60 °C, with a Kd of less than 100 µM and wherein said adsorbent material does not cause reduction or destruction of said corrin-containing compound. For the first time, an adsorbent material has been developed that is capable of binding corrin-containing compounds with high affinity to the £ site without causing reduction or destruction of said corrin-containing compound. In a further aspect, the present invention provides a method for producing an adsorbent material suitable for the purification of a corrin-containing compound, said method comprising the steps of: i) provide an activated matrix comprising cyanide, and ii) treat said matrix with a solution containing azide. Without being bound by a specific theory, the manner of chemical initiation and the characteristics of the functional group point to a tetrazole residue which is the functional group, that is, the active substance, in the adsorbent material. In a further aspect, the invention relates to a method for producing an adsorbent material, comprising attaching a tetrazole group to a matrix or initiating the formation of a tetrazole group in a matrix. The present invention further provides a method for purifying corrin-containing compounds from a solution, said method comprising exposing said solution to an adsorbent material of the invention. Thus, in a further aspect, the invention relates to a method for purifying a corrin-containing compound from a solution comprising the steps of: i) provide a solution comprising a compound containing corrin, ii) provide an adsorbent material of the invention in the form defined herein, iii) expose said solution to said adsorbent material wind, iv) to separate complexes of the corrin-containing compound bound to said adsorbent material from the solution components that were not adsorbed onto the adsorbent material, Cbl) optionally perform a washing step, and vi) elute said corrin-containing compounds from said adsorbent material. In a further aspect, the invention provides cobalamin purified by the method of the invention and compositions, such as pharmaceutical compositions or food compositions, comprising said purified cobalamin. DESCRIPTION OF THE DRAWINGS Figure 1. Structure of cobalamin. A cobalamin molecule (Cbl, vitamin B12) consists of three fragments: 1) the corrinoid ring with the central cobalt atom, which differs in its valence depending on the coordination state; 2) the nucleotide with the base coordinated in the lower axial position (alpha); 3) the group coordinated to cobalt in the upper axial position (beta). Different groups can be linked with varying α-affinity at the beta site of Cbl. Figure 2. Formation of tetrazole and its properties. Upper scheme. Reaction between CN-Br-activated sepharose and azide. The suggested scheme has two steps, where the initial attachment of azide to cyanide is followed by a cyclic transformation with the appearance of tetrazole. The reaction is given according to Ref. 11. Lower scheme. Tetrazole and its The groups are negatively charged at neutral pH and have a cap. potential city to attach two protons with pK a 4-5 and pK b (-3)-(-2) . Figure 3. Interaction between Cbl-OH2 and aminotetrazole. A) Spectrum of 50 µm Cbl-OH2 in the absence of tetrazole (solid line) and in the presence of 2 mM aminotetrazole (dashed line), 0.2 M phosphate buffer, pH 7.5, 37 °C. B) Change in absorbance of Cbl-OH2 after mixing with different concentrations of aminotetrazole (25-250 pM), conditions stated in A. The theoretical curves show the fit of the bi-molecular reaction Cbl + ATZ <-> Cbl-ATZ with k += 306 +20 M' and k.= (1.2 ±0.3) - 10" 3 S _1 . Figure 4. A) Adsorption of Cbl-OH2 onto the affinity adsorbent material followed over time. Cbl-OH solution 2 The mixture was combined with different volumes of specific adsorbent material Cbl. Final concentrations were: 50 / zM Cbl, 25-250 / zM of matrix-coupled functional groups. The reaction was carried out at pH 7.5, 37 °C and moderate stirring of the mixture. At certain intervals, 0.5 mL of sample was taken and quickly filtered. The amount of Cbl-OH2 in the solution was measured. The theoretical curves show the fit to the bi-molecular reaction Cbl + adsorbent material <-> Cbl - adsorbent material with k + = 55 + 6 M : S> and k_= (4.1 + 0.3) 10' 3 S' 1B) Equilibrium bond at 20 / zM Cbl-OH2 at different concentrations of adsorbent material. Incubation time was 5 hours and 22 hours (pH 6.0, 20 °C). The adjustment gave the following equilibrium dissociation constants: Ka = 2.8 / zM (5 hours) and Ka = 1.1 / zM (22 hours). Figure 5. Ideal pH for Cbl adsorption. The initial adsorption rate of Cbl-OH2 on the specific adsorbent material was plotted as a function of pH (20 °C). The final concentrations were 50 µM Cbl and 400 of the linking groups. The ideal pH was in the pH range of 3-7. Protonation-deprotonation of two groups with pKi 2.0 and pK 2 7.8 influenced the binding. Without adhering to any specific theory, comparison with known pK values ​​for the interacting molecules suggests the following conditions for efficient binding: 1) the tetrazole group of the adsorbent must be deprotonated (pH>2), 2) Cbl must be in the water form (Cbl-OH2, pH<7.5) but not in the hydroxyl form (C-bl-OH, pH>7.5). Figure 6. Binding of the Cbl-OH2 + Cbl-Ado mixture to the adsorbent. Binding of Cbls, which are typical for a natural source, was investigated at pH 7.5, 20 °C and different lighting conditions. The 50 µM mixture of Cbls had 20% Cbl-OH2 (weak £ group), 70% Cbl-Ado (strong 0 group, susceptible to light) and ~10% Cbl-CN (strong 3 group, resistant to moderate illumination). Adsorption conditions were: 1) darkness, 2) under diffuse light, 3) after 30 minutes of preliminary illumination with diffuse light. The remaining absorbance at t-> corresponded to 4% of Cbl-CN present as a contaminant in the Cbl-Ado preparation. Figure 7. Desorption of Cbl from the specific adsorbent material. Elution of Cbl from the adsorbent (0.1 mL) was conducted at 20 °C with 1 mL of the following solutions: 1) 10 mM KCN, pH 5.5 (open circles); 2) 10 mM KCN, pH 7.5 (triangle), pH 9.5 (square), pH 12 (rhomboid); 3) HCl IM (closed circles). Figure 8. Spectrum of eluted Cbls. After elution of Cbl with KCN (the product is Cbl-CN (Fig. 8A)) or 1M HCl (the product is Cbl-OH2 (Figure 8B)), the spectra of the preparations obtained were recorded and compared with the standards (solid lines). Dashed line corresponds to the preparations obtained immediately after elution (pH 7.5). Dashed lines show the spectrum of the final Cbl preparations (desalted, lyophilized and re-dissolved). Figure 9. Thin-layer chromatography of Cbl samples. A) Samples after adsorption and elution: 1 - Cbl-CN standard; 2 - Cbl-CN after hydrolysis with 1M HCl for 3 hours at 37 °C; 3 - Cbl-CN eluted from affinity material with 10 mM KCN, desalted and lyophilized; 4 - Cbl-CN produced from Cbl-OH2, which were eluted with HCl, desalted and lyophilized; 5 - Cbl-CN carboxy analogs. B) Purification of the Cbl model: 1 - Cbl-CN standard; 2 - purified Cbl-CN (Cbl-OH2 adsorbed from plasma, eluted with HCl, converted to Cbl-CN, desalted and lyophilized); 3 - Cbl-CN carboxy analogs. Figure 10. Conjugation of a tetrazole derivative, amino-tetrazole, via its amine with three different materials: Epoxy Sefarose 6B, CM Sefarose and Carboxy-Amberlite IRC76. DETAILED DESCRIPTION OF THE INVENTION Definitions In the form used here, the term "functional group" means the chemical fraction that interacts with the molecule being isolated (here, a molecule containing corrin) in such a way that the molecule being isolated remains attached to the adsorbent material under the conditions used for isolation. "Functional group" should not be interpreted as indicating that said group is the chemical group attached only to the matrix or that it is attached to the matrix in a specific way. For example, Figure 2 shows an example of an adsorbent material with tetrazole as a functional group, but other similar materials could be considered where, for example, tetrazole is linked to a matrix through different chemical fractions than those indicated in Figure 2, see for example Figure 10. The term "matrix" as used herein refers to a material, preferably a polymeric material, suitable for the binding (conjugation) or initiation of a functional group. The matrix may, for example, be in the form of a resin, a bead, a solid support, or another solid surface. In a preferred embodiment, the matrix is ​​an agarose-based resin. Preferred specific matrices are CNBr-activated Sefarose™, Epoxy Sefarose 6B, CM Sefarose, and Carboxy-Amberlite IRC76. Adsorbent materials of the invention and methods of preparation In one aspect, the invention provides an adsorbent material suitable for the purification of a corrin-containing compound, wherein said adsorbent material: It comprises a tetrazoline as a functional group. and / or - It is obtainable by a method that includes the following steps: i) provide an activated matrix comprising cyanide, and ii) treat said matrix with a solution containing azide, and / or - has a specific functional group £, wherein said functional group binds Cbl-OH2 at pH 3 to 7.5, at 5 °C to 60 °C, with a Ka of less than 100 gM and wherein said adsorbent material does not cause reduction or destruction of said corrin-containing compound. In a preferred embodiment, said corrin-containing compound is an aquo form of a corrin-containing compound or a form that is in equilibrium with an aquo form of a corrin-containing compound. In another preferred embodiment, said corrin-containing compound is cobalamin or an analogue thereof, more preferably aquo-cobalamin. The adsorbent material binds corrin-containing compounds with high affinity. In a preferred embodiment, said adsorbent material binds Cbl-OH2 apH3a7.5, a5°Ca 60 °C, with a K c from 0.01 to 100 gM, such as a Ka of 0.1 to 50 / zM, for example a Ka of 0.5 to 20 / zM, such as a Ka of 1 to 7 [M]. Ka is preferably determined in the manner described in Example 1. The adsorbent material, in a further preferred embodiment, has a maximum saturation of 0.1 to 500 mmol, such that such as 0.5 to 100 mmol, for example, 1 to 50 mmol, such as 4 to 8 mmol of the corrin-containing compound per liter of adsorbent material when packed in a column by gravity. In a further embodiment, the adsorbent material does not bind to Cbl-CN or Cbl-Ado. Many activated matrices known in the technology can be used as an activated matrix. A preferred activated matrix is ​​CNBr-activated agarose or CNBR-activated Sepharose. In a further aspect, the present invention provides a method for producing an adsorbent material comprising at least one functional group, said adsorbent material being suitable for the purification of a corrin-containing compound, said method comprising the steps of: i) provide an activated matrix comprising cyanide, and ii) treat the said matrix with a solution containing azide. Furthermore, in a further aspect, the invention relates to a method for producing an adsorbent material, comprising attaching a tetrazole group to a matrix or initiating the formation of a tetrazole group in a matrix; the tetrazole group can be attached to the matrix in any suitable manner, for example by means of any suitable substituent group on the carbon atom of tetrazole, as described in Example 4 and Figure 10. Methods for purifying compounds containing corrin The adsorbent material of the invention defined herein can be used for the purification of cor-rin-containing compounds from various sources, including biological sources. Thus, in another main aspect, the invention relates to a method for purifying a corrin-containing compound from a solution comprising the following steps: i) provide a solution comprising a compound containing corrin, ii) provide an adsorbent material of the form defined herein, iii) expose said solution to said adsorbent material, iv) to separate corrin-containing compound complexes bound to said adsorbent material from solution components that were not adsorbed to the adsorbent material, Cbl) optionally perform the washing step, and vi) elute said corrin-containing compounds from said adsorbent material. Compounds containing corrin are well known in the art and include, for example, corrinoids, Cbl synthesis intermediates, cobinamide, pseudo Bi2, factor A, and the like. In a preferred embodiment, said corrin-containing compound is an aquo form of a corrin-containing compound or a form that is in equilibrium with an aquo form of a corrin-containing compound. In another preferred embodiment, said corrin-containing compound is cobalamin or an analogue thereof, more preferably aquo-cobalamin. In some embodiments of the purification method, the said solution is exposed to light before and / or during step iii). This can be done, for example, to convert forms of Cbl that do not bind to the adsorbent material into forms that do bind to the material, such as Cbl-OH2. Furthermore, in some embodiments, the purification method comprises pre-treatment of the solution prior to step iii) to release corrin-containing compounds from complexes with other molecules, such as proteins. Such pre-treatment is preferably carried out by heating said solution, preferably at a low pH, such as pH 3. The exposure in step iii) of the purification method, in preferred embodiments, is carried out at a pH between 3 and 7. The purification method can be carried out by passing the solution containing corrin through a column packed with the adsorbent material of the invention. Thus, in some embodiments of the methods described above, steps iii) and iv) comprise passing said solution through a column comprising said adsorbent material. The elution of adsorbed molecules containing corrin can be done in several ways. In some embodiments, the elution step vi) comprises the use of cyanide (CN'). This is preferably carried out at pH 7-12, and is preferably followed by an e-step of conversion from the di-cyano form to the mono-cyano form, by For example, by acidification. In other embodiments, the elution in step vi) comprises the use of an acid, such as HCl, preferably followed by a neutralization step, and an additional conversion step to the cyano form by adding cyanide. The purification method preferably includes an additional step of purifying said compound containing coriander from salts contained in the sample. This can be accomplished in many ways, including dialysis, and / or adsorption using charcoal. During the use of charcoal, said purification is preferably carried out by adsorption of said corrin-containing compounds onto the charcoal followed by separation of corrin-containing compounds bound to the charcoal from solution components that do not adsorb onto the charcoal, and elution of the adsorbed corrin-containing compounds from the charcoal. Cobalamins and compositions of the invention In a further aspect, the invention relates to cobalamin obtained or obtainable by the purification method of the invention. In a further aspect, the invention relates to a composition comprising cobalamin obtained or obtainable by the purification method of the invention. In a further aspect, the invention relates to a pharmaceutical composition comprising cobalamin obtained or obtainable by the purification method of the invention and a pharmaceutically acceptable diluent, vehicle or excipient. vel . The compositions of the invention can be adapted for administration by any appropriate route, for example, by the oral route (including buccal or sublingual), topical route (including buccal, sublingual or transdermal), or parenteral route (including subcutaneous, intramuscular, intravenous or intradermal). Such formulations can be prepared by any method known in pharmaceutical technology, for example, by combining the active ingredient with the vehicle(s) or excipient(s). Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whipped creams; or oil-in-water or water-in-oil liquid emulsions. Pharmaceutical formulations adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. Pharmaceutical formulations adapted for topical administration in the mouth include diamond-shaped lozenges, pastilles, and mouthwashes. Pharmaceutical formulations adapted for parenteral administration include sterile aqueous and non-aqueous injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the target recipient; and sterile aqueous and non-aqueous suspensions that may include suspending agents and thickening agents. The formulations may be presented... in single-dose or multi-dose containers, for example, sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid vehicle, for example, water for injections, immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. Preferred unit-dose formulations are those that contain a daily dose or subdose, or an appropriate fraction thereof, of an active ingredient. It should be understood that, in addition to the ingredients specifically mentioned above, formulations may also include other conventional agents in the technology, taking into account the type of formulation in question; for example, those suitable for oral administration may include flavoring agents. In a further aspect, the present invention provides a food supplement comprising cobalamin purified by the method of the invention. Examples Example 1 1.1 Preparation of the adsorbent material CNBr-activated Sefarose 4B (Pharmacia) was packed into a column, thoroughly washed with 1 mM HCl, and then reacted with 0.02 M NaN3 in 0.1 M NaH2PO4 buffer (Pi-buffer), pH 7.5 at room temperature. Two volumes of the medium bed were pumped through the column for 1 time. The reaction between Sepharose and azide can also be carried out in batches, where 1 volume of the acid-washed matrix is ​​incubated for 1 hour with 2-3 volumes of Na N3 0.02 M in Pi-buffer 0.2 M, pH 7.5 with periodic stirring. After the coupling procedure, the adsorbent material was washed with Pi-buffer 0.1 M, pH 6.0-7.0 and then with water. The prepared material was stored at room temperature as an adsorbent material in the suspension : water (1:1). 1.2 Chemistry of the reaction The exact reaction mechanism between CNBr-activated sepharose and azide is not known. The scheme in Figure 2 suggests a possible mechanism according to relevant literature data. (11) . The reaction probably comprises two steps, wherein the first attachment of azide to Sepharose is followed by a cyclic transformation into a tetrazole group. In order to test this hypothesis, the binding of water-soluble starch-tetrazole in Cbl-OH2 (pH 7.5, 37 °C) was analyzed. The intention was to compare the rate constants of amino-tetrazole with those of NaN3 and those of the adsorbent material produced. Amino-tetrazole showed coordination capacity with the Cbl-13 site, as demonstrated by the spectral changes detected in Cbl-OH2 after mixing with amino-tetrazole (Figure 3a). The spectrum obtained was quite typical of Cbl-NR (6). The ability of tetrazole-containing compounds to bind to the Cbl p-site was previously unknown. The kinetics of this reaction were investigated when the transition of the Cbl-OH2 spectrum to Cbl-tetrazole was monitored in time. The relevant rate constants were calculated from the curves shown (Figure 3b). The results of a comparative assay of azide, amino-tetrazole and the adsorbent material of the invention are presented in Table 1. They support the theory that the functional group introduced into Sepharose is a tetrazole group. Table 1. Coordination rate constants for the p site of Cbl-OH2 measurements for c different compounds Reaction compound K + (M _1 s_1 ) K- í 3 - 1 Kd(pM) Conditions NaN3 2,500 0.13 50 3 7 °C, pH 7.5 Amino-tetrazolium 306 12 x 10" 4 3.9 3 7 °C, pH 7.5 Aminotetrazole 75 3.4 x 10" 4 2 0 °C, pH 7.5 Adsorbent material 55 4.1 x 10' 4 3 7 °C, pH 7.5 Adsorbent material 17 1, 1 x 10' 4 2 0 °C, pH 7.5 Adsorbent material 25 1.0 x 10" 4 20 °C, pH 7.5 Adsorbent material Equilibrium bond kd = 2.8 (5 h) : 1.1 (22 h) 20 °C, pH 7.5 Example 2. Properties of the specific adsorbent 1 of Cbl-OH2 2.1. Batch saturation of the activated azide matrix with Cbl for the highest possible level The binding of Cbl-OH2 in the produced adsorbent material was tested. Efficient saturation of the adsorbent material with Cbl was observed when 0.05 mL of the adsorbent material from the suspension:water (1:1) was mixed with 0.5 mL of Cbl-OH. 2 1 mM in 0.1 M Pi-buffer, pH 6, and incubated for 5 minutes. The sample was centrifuged (10,000 rpm, 30 s), and the colored supernatant was washed with 2 mL of 0.1 M Pi-buffer, pH 6, for 1 minute. The adsorbent material precipitated by centrifugation, and the bound Cbl was eluted from the pellet as described in section 2.2. The concentration of eluted Cbl was measured according to its absorbance spectrum. The concentration of Cbl binding groups in the packed adsorbent was estimated at 4-8 mM. The amount of active groups in the resin depended on the original Sepharose. Sepharose newly activated by CNBr showed a higher level of groups initiated by azide treatment. 2.2. Elution of Cbl from the adsorbent material in analytical tests Incubation of the adsorbent material saturated in Cbl (0.02-0.1 mL) in 1 mL of 2 mM KCN ​​at pH 7-9 and 95 °C for 5 minutes caused the complete release of the adsorbed Cbl in the form of dicyano, i.e., Cbl with CN groups coordinated to cobalt, both in the upper and lower axial positions (Cn-Cbl-CN). The eluted Cbl was quantified by its absorbance. It was easier to detect dicyano-Cbl, due to this form of the ligand having the highest extinction coefficient, compared to all other Cbls (6). 2.3. Saturation of the adsorbent material column without leakage. When a 0.2 mL column with adsorbent was subjected to a 100 pM flow of Cbl-OH2 at pH 6, it was possible to reach a concentration of 2 mM in the affinity material before any visual leakage. At a saturation level above 2 mM, the column eluate began to contain gradually increasing amounts of Cbl-OH2 (5-10 pM or more). Conversely, there was no perceptible leakage of the saturated adsorbent material to the same level when the applied solution contained no free Cbl-OH2. The adsorbed Cbl appeared susceptible to external Cbl-OH2, where the latter attacks the ligand in the adsorbent material and causes slight efflux at high saturation. 2.4. Kinetics of the interaction between the adsorbent and Cbl-OH2. The reaction between the adsorbent and Cbl was considered a reversible bimolecular process A + B → AB, which is expected to be valid at least to moderate saturation of the adsorbent material. In order to determine the kinetic parameters of the reaction, different amounts of the adsorbent material (corresponding to concentrations of 25-250 pM of the functional groups in the final suspension) were mixed with 50 pM Cbl-OH2. The process was carried out at both pH 7.5, 37 °C, 20 °C and pH 6.0, 20 °C. After incubation with slight agitation, 0.5 mL fractions of the suspension were rapidly filtered through a sponge packed in a Pasteur pipette. The concentration of Cbl in the filtered samples was measured and used to trace the Adsorption curves over time (Figure 4a). The experimental points were satisfactorily described by the rate constants shown in Table 1. Cbl binding in the material can be conducted at 5 °C or 60 °C, as well as at 20-37 °C, but with proportional deceleration / acceleration of the adsorption process. In another configuration, e-equilibrium binding was analyzed. Different amounts of the adsorbent material were incubated under gentle stirring with 1 mL of 20 pM Cbl-OH2 for 5-22 hours. The decrease in soluble Cbl-OH2 was plotted as a function of the concentration of functional groups in the suspension (Figure 4b). The equilibrium dissociation constants were then calculated (Table 1). Their values ​​were slightly lower than Ka calculated from the rate constants in the kinetic assay (Figure 4a).Comparison of the adsorption kinetics of Cbl-OH2 clearly indicates greater similarity of the specific Cbl adsorbent material to amino-tetrazole than to azide. This supports the hypothesis that the functional groups in azide-activated Sepharose are a tetrazole group, rather than a coupled azide group. The aforementioned kinetic experiment allowed some predictions to be made regarding the behavior of the adsorbent in the packed column. Thus, filtration of a medium containing Cbl-OH2 through an adsorbent material packed in a column (4 mM of tetrazolium groups presumed in the adsorbent material) will result in the adsorption of 95% of Cbl applied at a flow rate of two bed volumes for 1 minute or 99.8% at a flow rate of one bed for 1 minute (pH 6, ambient temperature). Experimental measurements corroborated the previous calculations and showed 95-96% adsorption of 2.5 pmol of Cbl filtered in a 1 mL column at a flow rate of 2 mL / min (pH 6, room temperature). Batch adsorption in the diluted suspension (1 volume of adsorbent material mixed with 99 volumes of a solution containing Cbl, 40 pM of presumed tetrazolo groups, 1 pM of Cbl in the final dilution, pH 6.0, 20 °C) gave approximately 80% adsorption of Cbl-OH2 after 30 minutes of incubation. 2.5. Dependence of adsorption on pH. The binding of Cbl-OH2 to a tetrazole group can be expected to be pH-sensitive, considering the numerous acid-base groups present in the reaction molecules: i) pK (-2) -(-1) for the second proton of the tetrazole, ii) pK 2-4 for the first proton in the tetrazole, iii) pK 0 for protonation of the Cbl nucleotide base, iv) pK 7.6 for acid-base equilibrium Cbl-OH + H +Cbl-OH2. Solutions with a wide pH range of 0-12 were tested as a medium for the adsorption of Cbl-OH2 onto the adsorbent material. Binding was performed in batches when 0.2 mL of the adsorbent material suspension was mixed with 1 mL of the medium containing 60 pM Cbl. The removal of Cbl from the supernatant was tracked over 1-30 minutes by optical measurements. The initial rate of binding was plotted as a function of pH (Figure 5). The graph shows that the best conditions are between pH 3 and pH 7. The approximate pK values ​​of the groups influencing binding were pK 2.0 and pK 7.8. As shown in Figure 5, no adsorption was detected at pH 0 (1M HCl). The absence of binding at a low pH was not caused by deterioration of the adsorbent material because adsorbent material washed with HCl recovered its ability to take up Cbl-OH2 from the solution as soon as the pH was adjusted to 4-7. The effect appeared to be induced by protonation of the adsorbent material at a low pH, as is known for tetrazole groups (11). Increasing the pH above 7 also caused a slowdown in bonding, presumably caused by the transition of aquo-Cbl to its hydroxyl form (in which the OH group has a higher affinity for the p-site of Cbl than water). However, work at pH 7.5 was not difficult compared to pH 6, as demonstrated by the data in Table 1. 2.6. Binding of biologically relevant Cbls with TZ-Sepharose The main forms of Cbl available from natural sources are Cbl-Ado, Cbl-Met, and Cbl-OH2, where the first two are light-sensitive. Therefore, the adsorption characteristics of a mixture of 20% Cbl-OH2 and 80% Cbl-Ado were tested under the following conditions: 0.1 mL of adsorbent material, 0.9 mL of buffer, 50 pM Cbl, pH 6, 20 °C. The samples were differentially illuminated: 1) kept in the dark, 2) incubated in diffuse ambient light during binding, 3) subjected to 30 minutes of diffuse illumination before binding. The results are represented as the remaining optical density in the solution as a function of incubation time (Figure 6). As expected, 20% Cbl-OH2 was rapidly removed from the medium of all samples. In contrast, the fact that Cbl- Ado remained different across three samples in the study. Thus, the initial phase of rapid adsorption was followed by a very slow binding of Cbl-Ado when the sample was kept in the dark. Moderate illumination of the suspension caused a notable acceleration of adsorption, whereas preliminary illumination of the solution made the binding rate practically indifferent for pure Cbl-OH2. It can be calculated that the photochemical conversion of Cbl-Ado to Cbl-OH2 was quite efficient, and practically all of the original Cbl-Ado group remains accumulated in the adsorbent material after 1 hour of incubation, even under moderate illumination. The remaining absorbance of the supernatant was not associated with either Cbl-Ado or Cbl-OH2, but originated from contaminant Cbl-CN found in the recorded spectrum (not shown). 2.7. Desorption of Cbl from the adsorbent material under different conditions Release of Cbl from adsorbent affinity material can be carried out in several ways depending on the purpose of the work. The method for rapid quantitative elution in 2-10 mM KCN, pH 7.5 at 95 °C is suitable for analytical purposes and is described earlier in section 2.2. Two other methods, which are more appropriate for large-scale Cbl purification, are presented below. The first method also employs 10 mM KCN, but desorption is performed under milder conditions. Thus, Cbl can be eluted at room temperature and pH 7-12, if the time if the incubation period is long enough (figure 7). Approximately- Approximately 10 hours were sufficient to remove 99% of the adsorbed Cbl from the adsorbent material. Since this reaction was irreversible, an excess of eluent was not actually required to release all the Cbl from the packed column. The ligand was obtained as CN-Cbl-CN, and the presence of cyanide at both coordination positions accelerated the release from the adsorbent material. Acidification of the medium to pH 2-3 caused a transition to the mono-cyano form (Cbl-CN, vitamin B). 12 ) in 20 minutes. The absorbance spectrum of the substance obtained did not differ from the standard Cbl-CN solution (Figure 8). The advantage of KCN elution is the transformation of Cbl into cyano and dicyano forms, which are quite stable with respect to different chemical reagents. However, the disadvantage of this method is the application of the hazardous compound KCN. Elution of adsorbed Cbl can also be carried out in 1M HCl, according to the pH dependence of Cbl binding (Figure 5). The process was rapid, and 90-95% elution was obtained after 1 minute of incubation of the bath in the acid (Figure 7) at a ratio of adsorbent material:acid = 1:10. Release of the remaining 5-10% of Cbl required fresh HCl, i.e., the attachment-detachment process in the acid was reversible, unlike the irreversible elution induced by KCN described above. During column desorption, it was sufficient to pump two volumes of 1M HCl beds through the adsorbent material (5 minutes per volume) in order to release 95% of the bound ligand. Complete recovery of the adsorbent can be achieved, if necessary, by washing with two additional volumes of acid. It is not recommended... It is difficult to maintain Cbl in IM HCl for a long time because of its slow hydrolysis with carboxy analogs. Therefore, the eluted sample was neutralized with 5 M NaOH and a buffer was added to maintain the pH at a desirable level (pH 5 or pH 7.5). Spectral measurements showed that the obtained sample contained Cbl-OH2 and the spectrum did not differ from the standard (Figure 8). The aqueous form of Cbl at pH 5 can be easily converted to the stable cyano form by adding KCN in a slight excess above Cbl. Conversion to Cbl-CN may be preferable due to the protective properties of cyanide. 2.8. Stability of the specific adsorbent material Cbl in a different medium The stability of the adsorbent material is important for its long-term application, and the prepared adsorbent proved to be resistant to degradation. For example, the adsorbent material can be stored in water or 1-10 mM NaN3 (antibacterial agent) at room temperature for one year without any visible deterioration. Not only storage, but also Cbl desorption conditions can potentially influence the properties of the adsorbent material. Therefore, we tested the effect of the elution medium on the adsorbent material. Tests showed that the adsorbent could be stored at room temperature in 10 mM KCN, 0.1 M Pi, pH 7.5 for at least two weeks without any loss of Cbl binding capacity. The maximum saturation and adsorption rate remained the same as in the fresh material. On the other hand, examination of the adsorbent material after incubation in 1M HCl was of higher priority because the pH of this solution was outside the stability region reported for Sefarose (pH 2-11). The test was performed as follows: the adsorbent material, after 30-50 hours of incubation in the acid, was subjected to a flow of a Cbl-OH2 solution (0.1 M Pi, pH 6.0, 200 pM Cbl). The optical density of the eluted liquid was measured, and the experiment ended as soon as the Cbl leakage reached 20 pM, that is, 10% of that in the applied medium. The amount of adsorbent ligand was then determined, and the data are presented as the concentration of Cbl in the adsorbent material (Table 2). Table 2. Cbl binding properties of the adsorbent material after different incubation times with HCl. 1M. Time in HCl Degree of saturation without leakage of Cbl in the flow: two volumes of tfl / minute - half volume of tfl / minute - 0 hours 2.5 mM 3.1 mM 30 hours 2.2 mM 2.4 mM 50 hours 1.2 mM 1.9 mM Incubation in 1M HCl caused some deterioration of the resin accompanied by prior leakage of Cbl and a lower degree of saturation. The effect was more visible under low-flow adsorption (two bed volumes per minute), but not as pronounced at the lowest flow (half the bed flow per minute). In any case, the column could be saturated at the level of 2 mmol of Cbl per liter of adsorbent material. A flow rate of 0.5 volume / minute was maintained even after 50 hours of incubation in 1M HCl. This result demonstrates that the specific Cbl material can potentially survive 300 adsorption-desorption cycles (allowing 10 minutes for each HCl elution) without being significantly damaged. 2.9. Desalination of the Cbl sample after elution of the adsorbent material. The preparation of Cbl from adsorbent affinity material cannot be used immediately after elution due to the high concentration of NaCl (HCl elution followed by NaOH neutralization) or the presence of 7-8 mM free KCN (KCN elution). The two methods described below are the most suitable for removing low molecular weight elements from the solution. The separation of Cbl from salts can be conducted using the difference in their mass (1300 for Cbl and < 100 for the salts). This can be done by dialysis against water using a membrane with pore diameters corresponding to the 500-1000 cutoff size. Several dilution-concentration cycles with a membrane with the same characteristics are also possible. The dialysis-based method worked well for Cbl-CN because no changes in the UV and visible spectrum were introduced compared to the standard. However, analysis of the final Cbl-OH2 preparation showed some distortions in the spectral shape, presumably due to the reaction of site 3 with contaminants. Thin-layer chromatography analysis showed the presence of a yellow-colored Cbl (10-20%), which drifted slightly. The concentration increased faster than the main fraction, but still overlapped with it (Figure 9A, lane 3). No substance was found if Cbl-OH2 was converted to Cbl-CN before dialysis and lyophilization (Figure 9A, lane 4). The presence of a secondary impurity in the preparation of concentrated Cbl-OH2 may suggest recrystallization of the sample by conventional methods. The Cbl sample can also be desalinated using the charcoal method. This procedure is not applicable to Cbl-OH2 due to the sensitivity of its p-site to some components in charcoal (the final Cbl preparation turned yellow and contained π groups (3 other than water)). On the other hand, Cbl-CN with its strongly associated π group is suitable for this type of treatment. In experiments, a maximum saturation of 20-25 pmol Cbl per 1 g of charcoal was obtained. Adsorption occurred instantaneously, and the solid fraction could be separated from the solution by gravitational sedimentation (2-3 hours), rapid centrifugation (1 minute 3000 g) or filtration. The pellet was washed with water, and the adsorbed Cbl was eluted with 96% ethanol in two portions (each incubation for 30 min). Example 3. Purification of the Cbl model 3.1. The source of Cbl A model system using an intrinsic factor-binding protein (IF) Cbl (5, 12) saturated with Cbl-OH2 and added to human plasma diluted 1:5 with 0.1 M citrate buffer pH 3.0 was established. The final concentration of IF-Cbl was 10 pM (- 0.01 mg / mL of Cbl) in a total volume 5 mL. The concentration of contaminating proteins was approximately 15 mg / mL. 3.2. Cbl Extraction The sample containing Cbl was heated to 95 °C for 30 minutes to release Cbl-OH2 from the protein complex. Treatment at pH 3 was accompanied by approximately 30% degradation of the ligand. For comparison, analogous treatment at pH 7 caused complete degradation of Cbl-OH2, possibly due to reduction with SH-containing compounds present in the plasma. The heated sample was cooled and neutralized with NaOH at pH 5.0, whereupon debris was removed by centrifugation. The pellet, which corresponded to 1 / 3 of the total volume, was then washed with 0.1 M citric acid buffer, pH 5.0, centrifuged, and both supernatants were pooled. The final sample volume was 6 mL. Other extraction methods may also potentially be applied. 3.3. Adsorption on TZ-Sepharose The ligand was adsorbed onto the specific adsorbent material Cbl (0.1 mL) packed in a Pasteur pipette at a flow rate of 0.2 mL / min (two bed volumes per minute). The adsorbent with bound Cbl was washed with 1 mL of 1 M NaCl, 0.1 M Tris, pH 7.5 and then with 1 mL of water. 3.4. Cbl Elution The adsorbed ligand was eluted from the column with 0.3 mL of 1M HCl, whereupon the solution was neutralized with 5M NaOH, and citrate buffer was added at a concentration of 0.1 M to bring the pH to ~3.0. The resulting Cbl-OH2 was converted... The sample was taken in Cbl-CN by adding 1 mM KCN ​​and heating the sample for 5 minutes at 95 °C. 3.5. Desalination and freeze-drying The Cbl salt solution was dialyzed against water. 5. Using a membrane with a molecular weight cutoff of 500, the sample was then lyophilized. Analysis of the dissolved preparation showed an absorbance spectrum virtually identical to the standard in both the UV and visible regions (Figure 8). Thin-layer chromatography (Figure 9B) revealed a colored fraction (lane 2) with a mobility that matched the standard (lane 1). The amount of purified Cbl corresponded to 60-65% of that added to the template sample. The main loss of 25-30% occurred during the extraction of Cbl-OH2 from the plasma. 15 Example 4. Conjugation of amino-tetrazole with different rentes matrices The active nature of the tetrazole group was confirmed in an independent experiment. Thus, the tetrazole derivative, amino-tetrazole, was conjugated via its amine 20 with three different materials: Epoxy Sefarose 6B, CM Sefarose, and carboxy-Amberlite IRC76 (Figure 10). As a result, the Cbl-inert materials (Epoxy- and CM-Sefaroses) acquired a Cbl binding capacity of 2 mM and 6 mM and behaved in all other respects identically to the matrix described previously. A low non-specific Cbl binding to Amberlite was accelerated by a factor of 10 after tetrazole attachment. 4.1 Experimental Details 10 mL of the tested matrix were washed with excess water and a slurry of the reacting compounds was prepared: i) 20 mL of 0.1 M aminotetrazole, pH 12.3, were mixed with 10 mL of Epoxy Sefarose 6B; or ii) 20 mL of 0.1 M aminotetrazole, 0.2 M N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide, pH 5.5, was mixed with 10 mL of CM Sefarose or Carboxy-Amberlite IRC76. The pH of the latter mixtures was monitored during the first hour of the reaction and maintained in the pH range of 5.0–5.5 by adding 5 M NaOH. Incubation continued at room temperature with gentle agitation for two days. Then, the activated matrices were washed with excess water and tested for Cbl binding capacity. CONCLUSIONS FROM THE EXAMPLES A specific Cbl group was successfully initiated in an insoluble material by treating CNBr-activated sepharose with sodium azide (NaN3). Without being tied to a specific theory, kinetic analysis strongly suggests that the active substance in the resulting adsorbent material was a tetrazole group, and not an azide group. The tetrazole group, both in solution and coupled to a carrier material, effectively binds Cbl to its β site because of coordination with cobalt. This property of tetrazole was previously unknown. The obtained material showed high affinity for Cbl-OH2 with K d = 1-7 pM at pH 6-7.5 (Table 1) and maximum saturation equal to 4-8 mmol of Cbl per liter of adsorbent. Saturation of 2-3 mM can be obtained with minimal leakage. The binding of Cbl-OH2 to the adsorbent material was observed over a wide pH range from 3 to pH 7. The capture of Cbl-OH2 from a solution was efficient both during column filtration and in batch filtration. The adsorbent material does not bind Cbls with strongly associated π groups, for example, Cbl-CN or the biological form Cbl-Ado. However, diffuse illumination of the latter form of Cbl caused conversion to Cbl-OH2 accompanied by efficient adsorption. Two elution methods using Cbl-saturated affinity material were developed. The first employed 10 mM KCN, with the final product obtained as pure Cbl-CN. The advantage of this method is the protection of the Cbl p-site from potential interaction with contaminating substances. The second technique used 1M HCl as an eluent and allowed the product to be obtained as Cbl-OH2, which contains 10-20% Cbl with p-groups without being water. This was caused by reaction with some contaminants during the concentration of the Cbl sample. The affinity adsorbent proved durable and withstood all the applied reagents equally well. The analysis showed the material's potential capacity to withstand 300 adsorption cycles followed by elution with 1M HCl. The model purification was performed using Cbl-OH2 attached to a specific protein intrinsic factor mixed with plasma proteins. The main purification steps were: 1) extraction at pH 3 and 95 °C, 2) adsorption onto the affinity material, 3) elution with 1M HCl, neutralization and conversion of Cbl-OH2 to Cbl-CN, 4) de-salinization. and lyophilization of the solution. The product obtained does not differ from the standard according to the absorbance spectrum and mobility in thin-layer chromatography. REFERENCES 1. Marsh EN (1999) Essays Biochem., 34, 139-154. 2. Battersby AR (1994) Science, 264, 1551-1557. 3. Mervyn L. (1971) In: Comprehensive Biochemistry, Eds.: Florkin M. , Stotz E.H., Elsvier, Amsterdam, London, New York, Volume 21 , pp.153-177. 4. Baik H.W., e Russel R.M. (1999) Annu. Rev. Nu-tr. , 19, 357-377. 5. Nexo E. (1998) In: Vitamin B12 e B12-Proteins, Eds.: Krautler B., Arigoni D., e Golding T. , Wiley-VCH, Weinheim, Germany, pp. 461-475. . 6. Pratt J. M. (1972) Inorganic Chemistry de Vitamin B12, Academic Press, London. 7. Gimsing P., e Nexo E. (1983) In: The Cobalamins, Ed.: Hall C.A., Churchill Livingstone, Edinburgh, pp. 7-30 . 8. Spalla C, Green A., Garofano L., e Ferni G. (1989) In: Biotechnology de Vitamins, Pigmets e Growth Fa-tors, Ed.: Vandamme E.J., Elsvier Applied Science, London, pp. 275-284. 9. Vandamme E.J. (1992) J. Chem. Tech. Biotechnol. 53, 313-327. 10. Firth R.A., Hill H.A.O., Pratt J. M. , Thorp RG, and Williams RJP (1969) J. Chem. Soc. (A), 381-386. 11. Koldobsky GI, and Ostrovsky CBI.A. (1988) Chemistry Heterocyclic Compounds (USSR) 5, 469-480. 12. Fedosov SN.. Berglund L., Fedosova N. , Nexo E., and Petersen TE (2002) J. Biol. Chem. 277 , 9989 – 9996 .

Claims

CLAIMS 1. Adsorbent material suitable for the purification of a corrin-containing compound, CHARACTERIZED in that said adsorbent material: comprises a tetrazoline as a functional group, and / or - It is obtainable through a method that includes the following steps: i) provide an activated matrix comprising cyanide, and ii) treat said matrix with a solution containing azide, and / or - has a specific functional group (3, wherein said functional group binds Cbl-OH2 at pH 3 to 7.5, at 5 °C to 60 °C, with a Kd of less than 100 pM, and wherein said adsorbent material does not cause reduction or destruction of said corrin-containing compound.

2. Adsorbent material, according to claim 1, CHARACTERIZED in that said corrin-containing compound is cobalamin or an analogue thereof.

3. Adsorbent material, according to any of the preceding claims, CHARACTERIZED in that said corrin-containing molecule is an aquo form of a corrin-containing compound, preferably aquo-cobalamin, or a form of a corrin-containing compound that is in e-equilibrium with the aquo form of a corrin-containing compound.

4. Adsorbent material, according to any of the preceding claims, CHARACTERIZED in that that said adsorbent material binds Cbl-OH2 at pH 3 to 7.5, at 5 °C to 60 °C, with a K d From 0.01 to 100 pM, such as a Kd from 0.1 to 50 pM, for example a Ka from 0.5 to 20 pM, such as a Kd from 1 to 7 pM.

5. Adsorbent material, according to any of the preceding claims, CHARACTERIZED in that said adsorbent material has a maximum saturation of 0.1 to 500 mmol, such as 0.5 to 100 mmol, for example 1 to 50 mmol, such as 4 to 8 mmol of the corrin-containing compound per liter of the adsorbent material when packed in a column by gravitational force.

6. Adsorbent material, according to any of the preceding claims, CHARACTERIZED in that said activated matrix is ​​CNBr-activated Sepharose.

7. A method for producing an adsorbent material, according to any of the preceding claims, CHARACTERIZED in that it comprises attaching a tetrazole group to a matrix or initiating the formation of a tetrazole group in a matrix.

8. Method, according to claim 7, CHARACTERIZED in that said matrix is ​​Epoxy Sepharose 6B, CM Sepharose or Carboxy-Amberlite IRC76.

9. Method for producing an adsorbent material according to any one of claims 1-6, CHARACTERIZED in that it comprises the steps of: i) provide an activated matrix comprising cia grandson, and ii) treat said matrix with a solution containing azido.

10. A method for purifying a corrin-containing compound from a solution, characterized in that it comprises the following steps: i) provide a solution comprising a compound containing corrin, ii) provide an adsorbent material in the form defined in any one of claims 1-6, iii) expose said solution to said adsorbent material, iv) to separate complexes of the corrin-containing compound bound to said adsorbent material from the solution components that were not adsorbed onto the adsorbent material, (v) optionally perform a washing step, and vi) elute said corrin-containing compounds from said adsorbent material.

11. Method according to claim 10, CHARACTERIZED in that said corrin-containing compound is cobalamin or an analogue thereof.

12. Method according to claim 10 or 11, CHARACTERIZED by the fact that said corrin-containing molecule is an aquo form of a corrin-containing compound, preferably aquo-cobalamin, or a form of a corrin-containing compound that is in equilibrium with the aquo form of a corrin-containing compound.

13. Method, according to any one of claims 10 to 12, CHARACTERIZED in that it comprises exposing said solution to light before and / or during step iii).

14. Method, according to any one of claims 10 to 13, CHARACTERIZED in that it comprises pre-treating said solution before step iii) to release corrin-containing compounds from complexes with other molecules, such as proteins.

15. Method according to claim 14, CHARACTERIZED in that said pretreatment is carried out by heating said solution.

16. Method, according to any one of claims 10 to 15, CHARACTERIZED in that the exposure in step iii) is carried out at a pH between 3 and 7.

17. Method, according to any one of claims 10 to 16, CHARACTERIZED in that steps ii-i) and iv) comprise passing said solution through a column comprising said adsorbent material.

18. Method, according to any one of claims 10 to 17, CHARACTERIZED in that step vi) comprises the use of a cyanide (CN') solution.

19. Method according to claim 18, CHARACTERIZED in that step vi) is carried out at pH 7-12.

20. Method according to claim 18 or 19, CHARACTERIZED by the fact that a conversion step to the mono-cyano form is carried out after step vi), for example, by acidification.

21. Method, according to any one of claims 10 to 17, CHARACTERIZED in that step vi) comprises the use of an acid, such as HCl.

22. Method according to claim 21, CHARACTERIZED in that a neutralization step is performed after step vi).

23. Method according to claim 21 or 22, characterized by the fact that an additional conversion step to the cyano form is performed by adding cyanide.

24. Method, according to any one of claims 10 to 23, CHARACTERIZED in that it comprises an additional step of purifying said corrin-containing compounds from the salts contained in the sample.

25. Method according to claim 24, CHARACTERIZED in that said purification is done by dialysis.

26. Method according to claim 24, CHARACTERIZED in that said purification is done by adsorbing said corrin-containing compounds onto charcoal followed by separating corrin-containing compounds bound to charcoal from the components of the solution that do not adsorb onto charcoal, and eluting the adsorbed corrin-containing compounds from the charcoal.