Method for purifying aromatic amino acids

Abstract

This invention relates to a method for purifying amino acids from an initial mixture, wherein the amino acids comprise an aromatic ring and have an acidity constant Ka, the method comprising: - a first step: contacting the initial mixture with a strong cation exchange resin at a pH greater than or equal to pKa; - a second step: eluting with an aqueous solution of an eluent with a pH greater than that of the first step, thereby allowing the collection of an amino acid-rich fluid.

Description

[0001] This application is a divisional application of the invention patent application with application number 2014102740320, application date June 18, 2014, and invention name “Method for purifying aromatic amino acids”. Technical Field

[0002] The present invention relates to a method for purifying aromatic amino acids (ie amino acids containing an aromatic ring, and primarily tryptophan, phenylalanine, tyrosine and histidine). Background Art

[0003] Aromatic amino acids can be produced fermentatively, enzymatically or via synthetic routes.

[0004] Tryptophan, phenylalanine and histidine (for infants) are members of the essential amino acid family: for example, they are used as nutritional supplements.

[0005] Amino acids, which must be prepared by fermentation, synthesis, enzymatic routes, contain impurities (sugars and residual enzymatic hydrolysates, minerals, pigments) that must be removed in order to achieve the purity required for their commercial use.

[0006] Various purification methods have been developed for the purification of aromatic amino acids. They are based on the following main techniques: crystallization, liquid / liquid extraction and binding on resins.

[0007] Purification by crystallization is carried out by aqueous or solvent routes. A pretreatment step before crystallization is sometimes provided in order to improve the purity of the crystals. This can be a purification step on activated carbon (documents: CN 101245047 and CN 102146052); a membrane filtration step on a stainless steel filter (document: CN 101376646); an ultrafiltration step (document: CN 101691349); or a combination of multiple continuous filtrations for removing various types of impurities, for example, a microfiltration / ultrafiltration / nanofiltration procedure (document: CN 01812009).

[0008] In order to obtain better purity, solvents such as acetic acid (JP 2097801 and EP 1 756 055) or aliphatic alcohols (EP 0 274 ​​728) are sometimes added during the crystallization process. The use of these solvents leads to increased production costs and costs for handling or recycling mother liquors.

[0009] The second purification path consists of liquid / liquid extraction of the amino acid. Therefore, the purification of tryptophan has been proposed, either by using a surfactant for forming a reverse micellar phase (document: CN 102382030), or by using toluene (document: EP 0 299 715). These techniques are by no means sufficient for obtaining high purity. Pretreatment and post-treatment steps are required. Moreover, the use of large volumes of solvents is economically uninteresting for the treatment of industrial volumes of amino acids at a scale of several thousand tons / year / unit.

[0010] Finally, a third route proposes treatment on polymer resins.

[0011] In documents CN 101812488, CN 101948420 and US 2013 / 0079527, the purification described is based on the capture and binding of the amino acid on an adsorptive (and therefore neutral) resin, followed by elution with ethanol (in the first two documents) or with salt (in the third document).

[0012] Document FR 2 581 654 describes the adsorption of tryptophan on a weakly crosslinked, strongly cationic polystyrene resin. The target compound is precipitated and then dissolved in an acid solution (especially a solution of sulfuric acid or hydrochloric acid at a pH of 1 to 2). The acid solution is then contacted with a cation exchange resin, washed with water and subsequently eluted with a base. This "batch" method is the route commonly used in industry today.

[0013] Document FR 1 502 814 describes the adsorption of tryptophan on a strong cationic resin or a strong anionic resin. In both cases, the resin selected has a low crosslinking rate. In the case of a strong cationic resin, the pH value of the mixture containing tryptophan is adjusted to 2 before contact with the resin. Elution is carried out in two stages: first with a neutral salt solution to remove the contaminating amino acids and then with an alkaline solution to desorb the tryptophan. This is a method that requires two different eluents and a large amount of recovery reagents.

[0014] The disadvantage of the above-mentioned treatment on resin is that a large amount of concentrated chemical recovery reagent (usually, 2 to 10% by mass) is required to elute the amino acid after binding. It also happens that the amount of effluent is equivalent to the amount of recovery reagent.

[0015] Document US 5,300,653 describes the batch separation of at least one aromatic amino acid from other non-aromatic amino acids on a single column on a gel-type strong cationic resin, which is first converted into the form of a salt with an alkali metal. The solution containing the amino acid preferably has a pH value of 2 to 12. When the solution passes through the resin, the amino acid is adsorbed and then desorbed with water without the need for any acid or base or organic solvent.

[0016] The disadvantage of this method is that this elution mode does not allow the control of the ionic type of the resin. In fact, the cationic species present in the solution to be treated can be exchanged with alkali metals without using any eluent capable of maintaining the initial ionic type. Separation operations between amino acids may be affected by these untimely ion exchange mechanisms: therefore, the yield and purity of the amino acids produced will run the risk of decreasing or changing over time in an uncontrolled manner.

[0017] Most of these treatments on resins are based on "batch" applications, i.e. non-continuous applications. The resin is arranged in a column and successive eluents (which must contain an amino acid; washing water; a recovered solution - a salt solution, a base solution, an acid solution or an alcohol solution) are filtered successively through the column.

[0018] However, the aforementioned document US 2013 / 0079527 describes a semi-continuous separation on an adsorption resin of the XAD7 type in a chromatography system having a simulated moving bed (SMB) with three separation zones. The system is eluted with water or with salt. The disadvantage of this method is that the operation is performed on an adsorption resin, which is therefore more expensive than an ion exchange resin. The adsorption resin also tends to be quickly contaminated. Its use requires a cleaning procedure with alcohol, alkali and acid to remove impurities that cause contamination, which increases the operating limitations for cost and installed equipment.

[0019] Therefore, there is a need to develop improved methods for purifying aromatic amino acids, particularly those capable of separating target amino acids from impurities such as salts, sugars, pigments with minimal consumption of chemicals. Summary of the invention

[0020] The present invention firstly relates to a method for purifying an amino acid from an initial mixture, wherein the amino acid contains an aromatic ring and has an acidity constant Ka, the method comprising:

[0021] - The first step: contacting the initial mixture with a strong cationic resin under the condition that the pH is greater than or equal to the pKa,

[0022] - Second step: elution is performed using an aqueous solution of an eluent having a pH value greater than that of the first step, so that an amino acid-rich fluid can be collected.

[0023] According to an embodiment, the initial mixture further comprises sugar and / or salt, and wherein the amino acid-enriched fluid comprises less sugar and / or salt than the initial mixture.

[0024] According to an embodiment, the second elution step is an isocratic elution step.

[0025] According to an embodiment, the amino acid has a basicity constant Kb and during the first step, the pH value is less than or equal to pKb-3, preferably less than or equal to pKb-4, most preferably less than or equal to pKb-5 and ideally less than or equal to pKb-6.

[0026] According to an embodiment, said second step of elution is performed directly after said first step of contacting without any intervening washing.

[0027] According to an embodiment, the eluent solution is a solution of a base in water, the base is preferably soda, potash, ammonia or a mixture thereof, and more preferably ammonia.

[0028] According to an embodiment, the concentration of the base in the eluent solution is 0.01 to 50 g / L, preferably 0.2 to 20 g / L, more preferably 0.5 to 10 g / L, and most preferably 1 to 5 g / L.

[0029] According to an embodiment, the ratio of the number of moles of base in the eluent solution used in the second step to the number of moles of amino acid in the initial mixture contacted in the first step is less than or equal to 3, preferably less than or equal to 2, preferably less than or equal to 1.5, preferably less than or equal to 1, preferably less than or equal to 0.9, preferably less than or equal to 0.8, preferably less than or equal to 0.7, preferably less than or equal to 0.6, preferably less than or equal to 0.5, preferably less than or equal to 0.4.

[0030] According to an embodiment, the amino acid is tryptophan or phenylalanine or tyrosine or histidine.

[0031] According to an embodiment, the strong cationic resin is a polystyrene-divinylbenzene copolymer.

[0032] According to an embodiment, the cross-linking rate of the strong cationic resin is 5 to 12%, preferably 6 to 11%, more preferably 7 to 10%, preferably 8 to 9%.

[0033] According to an embodiment, the strong cationic resin is in the form of a monovalent cation, preferably Na + or K + or NH4 + form, and most preferably NH4 + form.

[0034] According to an embodiment, the method is carried out in a multi-column chromatography device, which preferably comprises at most 30 columns, more preferably at most 20 columns, or at most 12 columns, or at most 8 columns, or at most 6 columns.

[0035] According to an embodiment, the method is implemented in a chromatography apparatus with a non-fixed bed, and is preferably implemented in a chromatography apparatus with a simulated moving bed, or a real moving bed, or an improved simulated moving bed, or a sequential simulated moving bed, and is preferably implemented in a chromatography apparatus with a sequential simulated moving bed.

[0036] According to an embodiment, the initial mixture is obtained at the end of the treatment of a raw material mixture, the raw material mixture is preferably a fermentation slurry (must), a synthetic slurry or an enzymatic slurry, and the treatment preferably includes one or more steps selected from centrifugation, pre-filtration, crystallization, tangential filtration, microfiltration and ultrafiltration.

[0037] According to an embodiment, the method comprises one or more steps for concentrating the amino acid-rich fluid, preferably chosen from concentration by evaporation (and in particular by evaporation with falling film), concentration by multiple-effect evaporation, membrane concentration and in particular nanofiltration.

[0038] According to an embodiment, at the end of the concentration step, on the one hand a concentrated amino acid fluid is collected and on the other hand a fluid of an eluent solution is collected, the method comprising repeatedly circulating this fluid or solution towards a second elution step.

[0039] According to an embodiment, the difference between the pH value of the eluent solution used in the second step and the pH value of the first step is greater than or equal to 2, preferably greater than or equal to 4, more preferably greater than or equal to 6, and especially greater than or equal to 8.

[0040] The present invention overcomes the disadvantages of the prior art. More specifically, an improved method for purifying aromatic amino acids is provided, in particular separating the target amino acid from impurities (eg, salts, sugars, pigments) with minimal consumption of chemicals.

[0041] This can be achieved by using strong cationic resins (ion exchange resins), which are not used in ion exchange mode. The present invention is based on a chromatographic elution mode, in which the aromatic amino acids are separated from their initial solution by a retardation phenomenon caused by their affinity for the resin. Therefore, the present invention intends to bring the amino acids into contact with the resin mostly in the form of zwitterions (pH greater than or equal to the pKa of the amino acid) and then elute them with a solution having a higher pH value.

[0042] This mode of operation is different from the existing methods which implement the capture phenomenon by immobilization of amino acids and then desorb using a concentrated recovery eluent.

[0043] Without being bound by any theory, the inventors estimate that the separation mechanism employed may be an adsorption phenomenon caused by hydrophobic interactions between the π bonds of the aromatic groups present in the rings of the amino acids and / or between the π bonds of the doublet nitrogen atoms, as well as the hydrophobic effect of the resin matrix: the separation is therefore not constituted by a separation caused by ion exchange.

[0044] The invention can be practiced particularly with dilute eluent solutions and at lower eluent / amino acid ratios, which minimize chemical consumption and the amount of effluent.

[0045] The present invention also allows the implementation of a purification mechanism in a controlled manner, avoiding substantial variation in the yield and purity of the amino acid produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The diagram shows an apparatus capable of executing the method according to the present invention.

[0047] Figure 2 The time-dependent changes in tryptophan concentration (g / L, marked ○), conductivity (mS / cm, marked ×) and dry matter (Brix, marked ■) according to the volume injected into the chromatographic separation column (abscissa, BV or bed volume, i.e., multiple bed volumes) are shown. The operating conditions are specifically described in Example 1.

[0048] Figure 3 The time-dependent changes in tryptophan concentration (g / L, left scale, marked ○), conductivity (mS / cm, left scale, marked ×) and dry matter content (Brix, right scale, marked ■) on multiple chromatography columns (abscissa, number of columns) in a SSMB-type purification system are shown. The operating conditions are specifically described in Example 2.

[0049] Figure 4 The time-dependent changes of phenylalanine concentration (g / L, marked ○), conductivity (mS / cm, marked ×) and dry matter content (Brix, marked ■) according to the volume injected into the chromatographic separation column (abscissa, BV) are shown. The operating conditions are specifically described in Example 3.

[0050] Figure 5 The time-dependent changes of histidine concentration (g / L, marked ○), conductivity (mS / cm, marked ×) and dry matter content (Brix, marked ■) according to the volume injected into the chromatographic separation column (abscissa, BV) are shown. The operating conditions are specifically described in Example 4. DETAILED DESCRIPTION

[0051] The present invention will be described in more detail and without limitation in the following description.

[0052] The present invention allows to collect in purified form the aromatic amino acids present in the initial mixture, which is usually an aqueous solution (fermentation, synthetic slurry or enzymatically derived plug) containing various impurities (pigments, salts / minerals, sugars).

[0053] Therefore, according to an embodiment, the initial mixture contains sugars, for example, fermentation nutrients (glucose / sucrose, etc.) and / or metabolites. The total sugar content (and in particular the amount of monosaccharides and disaccharides) in the initial mixture can therefore be, for example, 0.01 to 100 g / L and preferably 0.1 to 15 g / L.

[0054] According to an embodiment, the initial mixture comprises salts, which may be mineral salts and / or organic salts. The total amount of salt in the initial mixture may thus be, for example, 0.01 to 80 g / L and preferably 0.5 to 20 g / L.

[0055] The conductivity of the initial mixture may be, for example, 1 to 200 mS / cm, preferably 2 to 100 mS / cm, and in particular 5 to 50 mS / cm.

[0056] According to an embodiment, the initial mixture is obtained at the end of the pretreatment by clarifying the culture fluid (or enzymatically). This clarification operation can be implemented using a variety of techniques:

[0057] - Decantation / centrifugation

[0058] - Tangential filtration on organic planar or spiral membranes having a cut-off threshold typically comprised between 2,000 and 300,000 Da.

[0059] - Tangential filtration on ceramic membranes having a cut-off threshold typically greater than 10,000 Da.

[0060] - Filtration on stainless steel filter.

[0061] - The combination and order of the above operations can be adjusted according to the conditions determined by the nature of the initial mixture and the purpose of the method.

[0062] The aromatic amino acid is preferably phenylalanine, tryptophan, tyrosine, histidine or a derivative thereof. It is also desirable to recover a mixture of aromatic amino acids.

[0063] The present invention provides chromatographic separation on strong cationic resins."Strong cationic resins"refers to resins with grafted acid functional groups (strong acids).

[0064] According to a preferred embodiment, "strong cationic resin" refers to a resin comprising sulfuric acid functional groups.

[0065] According to an embodiment, the cationic resin is a strong polystyrene-divinylbenzene (DVB) resin.

[0066] According to an embodiment, the resin is in the form of particles, the average volume diameter (Dv50) of which is preferably 20 to 600 μm, in particular 200 to 350 μm. Preferably, the distribution of the volume diameter of the resin particles has only one peak (monodisperse distribution). The uniformity coefficient of this distribution is, for example, less than or equal to 1.5, or less than or equal to 1.3, or less than or equal to 1.15. The resin particle size distribution can be determined by laser granulometry (NF 13320 standard).

[0067] The resin is preferably a macro-crosslinked resin (and thus not a gel-type resin). Therefore, according to an embodiment, the crosslinking rate (DVB rate, when the resin is the above copolymer) of the resin is 5 to 6%; or 6 to 7%; or 7 to 8%; or 8 to 9%; or 9 to 10%; or 10 to 11%; or 11 to 12%. The crosslinking rate is defined as the ratio of the amount of crosslinking agent monomer (e.g., DVB monomer) to the total amount of monomers applied in the production resin (e.g., DVB monomer and other resin monomers) (the amount is expressed in moles).

[0068] According to an embodiment, the resin is in a monovalent form. For example, it may be Na + , K + Or preferably NH4 + form.

[0069] Optionally, the process of the invention may include a prior step, provided that the resin is in the desired ionic form, particularly Na + , K + Form or preferably NH4 + The preceding step can in particular be carried out by passing a suitable salt solution or alkaline solution through the resin.

[0070] Amino acids behave like weak acids and weak bases. They have an acidity constant, Ka, and a basicity constant, Kb. The inventors defined pKa = -log 10 (Ka) and pKb = -log 10 (Kb). The pKa and the pKb represent the half-dissociation pH, that is, at this pH, the acid functional group and the base functional group of the amino acid are half-ionized, respectively.

[0071] In the amino acid, both the acid functional group and the amine (base) functional group are ionized, and this form of amino acid is called a zwitterion.

[0072] The pKa and pKb values ​​of the above four aromatic amino acids are as follows:

[0073] -Phenylalanine: pKa = 1.8 and pKb = 9.1.

[0074] -Tryptophan: pKa = 2.4 and pKb = 9.4.

[0075] -Tyrosine: pKa = 2.2 and pKb = 9.1.

[0076] - Histidine: pKa = 1.8 and pKb = 9.2.

[0077] The present invention provides the steps of contacting the initial mixture (either feed material or feed product) with the chromatography resin, and then the step of eluting.

[0078] In the first step, to ensure that the amino acid is effectively in zwitterionic form, the pH is greater than or equal to the pKa of the desired aromatic amino acid (or, if multiple aromatic amino acids are present, greater than or equal to the multiple pKas of the desired multiple aromatic amino acids).

[0079] According to certain embodiments, in the first step the pH is greater than or equal to pKa+0.5; or greater than or equal to pKa+1; or greater than or equal to pKa+1.5; or greater than or equal to pKa+2; or greater than or equal to pKa+2.5; or greater than or equal to pKa+3.

[0080] It should be noted that in the first step, the pH may also be less than or equal to pKa+3; or less than or equal to pKa+2.5; or less than or equal to pKa+2; or less than or equal to pKa+1.5; or less than or equal to pKa+1; or less than or equal to pKa+0.5.

[0081] According to certain embodiments, in the first step, pKa is less than or equal to pKb-3; or less than or equal to pKb-3.5; or less than or equal to pKb-4; or less than or equal to pKb-4.5; or less than or equal to pKb-5; or less than or equal to pKb-5.5; or less than or equal to pKb-6.

[0082] For example, in the first step the pH may be comprised between pKa and pKb-3; or between pKa and pKb-4; or between pKa and pKb-5; or between pKa and pKb-6.

[0083] According to certain embodiments, in the first step, the pH is between 1.8 and 1.9; or between 1.9 and 2.0; or between 2.0 and 2.1; or between 2.1 and 2.2; or between 2.2 and 2.3; or between 2.3 and 2.4; or between 2.4 and 2.5; or between 2.5 and 2.6; or between 2.6 and 2.7; or between 2.7 and 2.8; or between 2.8 and 2.9; or between 2.9 and 3.0; or between 3.0 and 3.2; or between 3.2 and 3.4; or between 3.4 and 3.6; or between 3.6 and 3.8; or between 3.8 and 4.0; or between 4.0 and 4.5; or between 4.5 and 5.0; or between 5.0 and 5.5; or between 5.5 and 6.0; or between 6.0 and 6.5; or between 6.5 and 7.0.

[0084] The pH of the feed material (initial mixture) can be adjusted by adding an acid or a base before contacting it with the resin.

[0085] In the second step, the eluent is an aqueous solution with a pH greater than that of the first step. The eluent can be an alkaline aqueous solution, such as, in particular, NH3 or NaOH solution.

[0086] The difference in pH (ΔpH) between the second step and the first step may be from 0.5 to 1.0; or from 1.0 to 1.5; or from 1.5 to 2.0; or from 2.0 to 2.5; or from 2.5 to 3.0; or from 3.0 to 3.5; or from 3.5 to 4.0; or from 4.0 to 4.5; or from 4.5 to 5.0; or from 5.0 to 5.5; or from 5.5 to 6.0; or from 6.0 to 6.5; or from 6.5 to 7.0; or from 7.0 to 7.5; or from 7.5 to 8.0; or from 8.0 to 8.5; or from 8.5 to 9.0; or from 9.0 to 9.5; or from 9.5 to 10.0; or from 10.0 to 10.5; or from 10.5 to 11.0; or from 11.0 to 11.5; or from 11.5 to 12.0.

[0087] The pH of the eluent (second step) may in particular be 7.0 to 7.5; or 7.5 to 8.0; or 8.0 to 8.5; or 8.5 to 9.0; or 9.0 to 9.5; or 9.5 to 10.0; or 10.0 to 10.5; or 10.5 to 11.0; or 11.0 to 11.5; or 11.5 to 12.0; or 12.0 to 12.5; or 12.5 to 13.0; or 13.0 to 13.5; or 13.5 to 14.0.

[0088] The eluent may be an alkaline solution, for example, NaOH or NH3, in water at a concentration of 0.001 to 0.01 g / L; or 0.01 to 0.1 g / L; or 0.1 to 0.5 g / L; or 0.5 to 1 g / L; or 1 to 2 g / L; or 2 to 3 g / L; or 3 to 4 g / L; or 4 to 5 g / L; or 5 to 6 g / L; or 6 to 7 g / L; or 7 to 8 g / L; or 8 to 9 g / L; or 9 to 10 g / L; or 10 to 15 g / L; or 15 to 20 g / L; or 20 to 25 g / L; or 25 to 50 g / L; or 50 to 100 g / L.

[0089] According to an embodiment, the number of moles of base used for elution, relative to the number of moles of amino acid injected, is less than or equal to 3; or less than or equal to 2; or less than or equal to 1.5; or less than or equal to 1; or less than or equal to 0.9; or less than or equal to 0.8; or less than or equal to 0.7; or less than or equal to 0.6; or less than or equal to 0.5; or less than or equal to 0.4.

[0090] The elution is preferably isocratic, ie the composition of the eluent does not change over time.

[0091] In a preferred embodiment, the cation of the base used for elution is the cation of the resin. Thus, for example, if the resin is Na + In the form of K, the eluent may be a solution of NaOH; if the resin is K + In the form of, the eluent may be a solution of KOH; and if the resin is NH4 + In the form of, the eluent may be a solution of ammonia.

[0092] Elution by alkali offers the possibility of maintaining the ionic form of the resin, which might otherwise be altered by mineral salts present in the feed stock solution. Moreover, without wishing to be bound by any theory, the applicants postulate an osmotic charge effect on the resin, which also tends to release adsorbed species.

[0093] The desired aromatic amino acids have a stronger affinity for the resin than most other impurities. Thus, the impurities (e.g., sugars and salts) are recovered in the raffinate fraction while the amino acids are recovered in the extract.

[0094] According to an embodiment, based on the total mass of sugars (especially monosaccharides and disaccharides) in the initial mixture, the total mass of sugars in the extract is reduced by at least 80%, preferably by at least 90%, or by at least 95%, or by at least 98%, or by at least 99%, or by at least 99.5%, or by at least 99.9%. Preferably, the extract (amino acid-rich fluid) does not contain or substantially does not contain any sugars (especially monosaccharides and disaccharides).

[0095] According to an embodiment, the total mass of salt in the extract is reduced by at least 80%, preferably by at least 90%, or by at least 95%, or by at least 98%, or by at least 99%, or by at least 99.5%, or by at least 99.9%, based on the total mass of salt in the initial mixture. Preferably, the extract (amino acid-rich fluid) does not contain or substantially does not contain any salt.

[0096] Depending on the embodiment, certain highly retained impurities may be desorbed by additional elution with water, brine, or a polar eluent.

[0097] According to an embodiment, there is no intervening wash between the contacting step and the elution step.The injection of the eluent thus directly follows the injection of the initial mixture.

[0098] According to an embodiment, the recovered amino acids from the plug extracted from the chromatographic separation are subjected to additional processing, such as:

[0099] - Decolorization on carbon.

[0100] - Demineralization.

[0101] - Concentration on reverse osmosis membranes or nanofiltration.

[0102] - Evaporation or multiple-effect evaporation, which optimizes energy consumption.

[0103] -crystallization.

[0104] - A combination of the aforementioned operations. The order can be adjusted according to the circumstances determined by the nature of the initial mixture and the purpose of the process.

[0105] According to a preferred embodiment, the eluent, preferably NH 3 solution, is separated from the amino acid for recycling at the very beginning of the chromatographic separation process and regenerated during a preceding work-up step.

[0106] The process according to the invention can be discontinuous ("batch process"), semi-continuous or continuous. Preferably it is semi-continuous or continuous.

[0107] The chromatographic separation according to the invention can be carried out in a chromatography unit with a fixed bed or preferably in a chromatography unit with a non-fixed bed.

[0108] In a chromatography system with a fixed bed, a mixture of compounds to be separated is filtered in a conventional cylindrical housing (or column). The column comprises a bed of porous material (stationary phase) that is permeable to the fluid. The leakage rate of each compound in the mixture depends on the physical properties of the compound. Most of the residual compounds on the stationary phase filter slower than the fewer residual compounds on the stationary phase. This principle provides the possibility of implementing the separation.

[0109] This treatment can be carried out in multiple columns connected in series or in parallel, but usually a chromatographic separation system with a fixed bed is applied with a single column.

[0110] Examples of such chromatography systems with a fixed bed are HPLC (High Performance Liquid Chromatography) or CYCLOJET TM (Recirculation system in static state) system.

[0111] The CYCLOJET TM The system is described in document US 6,063,284, to which particular reference is made. This is a discontinuous chromatographic separation system with a single column, in which the majority residue (i) and subsequently the smaller residue (ii) are separately collected at the outlet of the column, the unseparated part of the chromatogram is circulated by a main pump, and the mixture to be separated is periodically injected by means of an injection loop arranged substantially in the middle of the chromatographic recirculation part. After a number of chromatographic cycles, the process reaches a periodic steady state in which the amount of injected product is equal to the amount of separated product separately collected at the outlet of the column.

[0112] The document US 5,630,943 describes a CYCLOJET system using two columns. TM Optional method, which is provided as a special reference.

[0113] Systems with non-fixed beds are multi-column systems in which the relative position of the stationary phase bed and the relative positions of the injection and collection points of the fluids move over time.

[0114] Examples of such chromatography systems with a non-fixed bed are SMB (simulated moving bed), iSMB (improved simulated moving bed), SSMB (sequential simulated moving bed), AMB (actual moving bed), VARICOL TM 、MODICON TM 、POWERFEED TM , MCSGP or GSSR (multi-column gradient chromatography) system.

[0115] The SMB system comprises a plurality of separate columns containing adsorbents, which are connected in series. An eluent fluid passes through the plurality of columns along a first direction. The injection points of the feed fluid and the eluent fluid and the points for collecting the separated compounds are periodically and simultaneously switched by a set of valves. The overall effect simulates the operation of a single column comprising a moving bed of solid adsorbent, which moves in a counter-flow direction relative to the eluent fluid. Therefore, the SMB system consists of columns comprising a fixed bed of solid adsorbent through which the eluent passes, but the operation simulates a continuous moving bed as a counter-flow.

[0116] The most common form of the SMB system is an SMB system with four zones. Other possible forms are an SMB system with three zones and an SMB system with two zones (as described in the following paper: Two Section Simulated Moving Bed Process, Kwangnam Lee, Separation Science and Technology 35(4):519-534, 2000, which is specifically referenced).

[0117] In the iSMB and SSMB systems, there is at least one step where the system operates in a closed loop without any product inlet or outlet.

[0118] The iSMB system is described in documents EP 0 342 629 and US 5,064,539, to which specific reference is made.

[0119] The SSMB system divides the introduction and collection of fluids into sub-procedures that are performed periodically.

[0120] Other options for the SMB system are: as described in US 5,102,553 and the paper PowerFeed operation of simulated moving bed units: changing flow-rates during the switching interval (Zhang et al. Journal of Chromatography A, 1006: 87-99, 2003), the SMB system and POWERFEED that change with time TM system, which is specifically referred to as MODICON as described in US 7,479,228 TM system, to which specific reference is made; and, a SMB system with internal recirculation as described in document US Pat. No. 8,282,831, to which specific reference is made.

[0121] The AMB system has an operation similar to that of the SMB system. However, instead of switching the feed fluid and eluent injection points, and the collection points, a set of adsorption units (columns) are physically switched relative to the feed and collection points by a set of valves. Furthermore, the operation allows for the simulation of a continuous counter-flow moving bed.

[0122] VARICOL TM Chromatographic systems are described in documents US 6,136,198, US 6,375,839, US 6,413,419 and US 6,712,973, to which specific reference is made. VARICOL TM The system comprises a plurality of individual columns containing adsorbent, which are connected in series. The eluent flows into the columns in a first direction, and unlike the SMB system, the injection points of the mixture to be separated and the injection points of the eluent and the point of collecting the separated compounds are periodically switched asynchronously by a set of valves. The overall effect is to produce a separation zone with variable length over time, thereby dynamically configuring the stationary phase in the most useful area and obtaining a separation with fewer chromatographic separation units of similar power and an increased yield. Unlike the SMB system, VARICOL TM The system does not simulate the operation of a single column comprising a moving bed of solid adsorbent moving in a counter-flow direction relative to the eluent flow, so VARICOL TM The operating principle cannot be implemented in an equivalent AMB system.

[0123] A preferred embodiment of a simulated moving bed is shown schematically in the form of a sequential simulated moving bed (SSMB) chromatography system over six chambers or columns. Figure 1 This system operates according to a cyclic operation in four stages.

[0124] - Phase No. 1 (part A in the figure): loop phase, in which continuous circulation in a closed loop is maintained on all chambers arranged in series to switch the interstitial volume of one chamber to the next without injecting any eluent.

[0125] - Phase No. 2 (part B in the figure): Feed material / feed material injection. Feed material (F) is injected at the top of the fourth chamber. At the same time, substantially the same volume of residual liquid (R) is collected at the outlet of the fifth chamber. Chambers 4 and 5 form region 3 here. Chambers 2 and 3 form a separation region between the extraction liquid and the feed material injection. They form region 2 here.

[0126] - Phase No. 3 (part B in the figure): elution of the extract. The eluent (EL) is injected on the first chamber for eluting the extract (EX), and the eluted extract (EX) is collected at the bottom of the first chamber in substantially the same volume. Chamber 1 here forms zone 1.

[0127] - Stages 2 and 3 are preferably performed simultaneously to increase the productivity of the system.

[0128] - Phase No. 4: Elution of the raffinate. The eluent (EL) is injected at the top of the first chamber and the raffinate (R) is connected to the outlet of the fifth chamber in substantially the same volume. Chamber 6 is here a buffer chamber providing the possibility of ensuring separation between the rear end of the extract and the top end of the raffinate. It forms zone 4. This zone can be omitted in cases where the purity and / or the required yield are relatively limited.

[0129] These stages are operated in this order in the preferred embodiment, from 1 to 4. Their sequence forms a complete sequence.

[0130] The sequence (Stages 1 to 4) of switching chamber inlets and outlets is repeated six times by increasing the number of chambers in the system (from left to right): the feed material is thus injected at the top of chamber 1, sequence number 1, then at the top of chamber 2, sequence number 2, and so on.

[0131] A complete production cycle is achieved after completing six consecutive sequences, when the feed material injection point (initially at the inlet of chamber 1) is returned again to the inlet of the first chamber.

[0132] In the foregoing, a description of the SSMB system is given for the case where the chamber corresponds to a column. This is not restrictive, and the present invention is also applicable to a system in which the chamber or an additional partition cavity is a column part.

[0133] Furthermore, the number of columns present in zones 1, 2, 3 and 4 can vary depending on the desired separation quality. It is thus possible to design the same type of system with one chamber, two chambers, three chambers, four chambers and up to more than twelve chambers.

[0134] The method can further be applied in a discontinuous multi-column device with a closed loop or an open loop, such as the DCC system described in patent document WO 2007 / 012750.

[0135] According to an embodiment, a multi-column device (particularly of SMB type, etc.) comprises one or more cleaning zones that disconnect the columns of the separation circuit to periodically desorb strongly adsorbed compounds (impurities) and then regenerate and rebalance the adsorbent with a separate eluent.

[0136] Example

[0137] The following examples illustrate the invention without limiting it.

[0138] Example 1 - Tryptophan Retention Studies

[0139] In this example, tryptophan fermentation slurry was treated by adjusting pH = 2.6 followed by pretreatment by microfiltration and diafiltration on ceramic membranes.

[0140] Products XA2014-22 resin was used for treatment, with a volume of 30 mL / column (in this example, 30 mL represents 1 BV, where BV refers to the bed volume of the resin). The resin is a strong cationic resin of the polymerized polystyrene-DVB type with 8% DVB, a monodisperse particle size of 220 μm, and an ion exchange capacity of 1.8 equivalents / L 树脂 The resin was previously converted to the Na form by means of a 4% soda solution.

[0141] The initial mixture for the chromatography step contained 17.5 g / L tryptophan, 3.7 g / L residual sugars, and a conductivity of 18.5 mS / cm induced by the salts in solution.

[0142] This mixture is injected in excess onto the resin. During the injection phase, different substances of different nature are observed on the resin:

[0143] - Salt-containing impurities are not retained on the resin: it can be estimated that ionic repulsion phenomena between their ionic charge and that of the resin prevent them from penetrating into the matrix. The conductivity measured at the column outlet during the elution phase is identical to that of the initial mixture.

[0144] -Sugars, neutral substances, can penetrate into the matrix of the resin and are gradually slowed down but are not retained: they co-elute with the salts during the injection phase.

[0145] - Most of the tryptophan injected at pH = 2.6 in the form of zwitterions experiences a delay phenomenon due to the interaction between its aromatic groups and the resin groups.

[0146] Elution of the thus retained tryptophan was obtained with a soda concentration of 4 g / L at 2 BV / h.

[0147] Figure 2 The time-dependent variation of the concentration in a point in the system is illustrated, where the entire process of the separation step is seen, starting with the elution of the raffinate and ending with the washing of the extract. Stage A is the injection phase of the initial mixture, stage B is the elution phase with the soda solution.

[0148] This system provides the following operating possibilities:

[0149] - 205.6 g of tryptophan per liter of resin was processed in an 8-hour preparation cycle;

[0150] - During collection of the tryptophan extract, the concentration was <100 μS / cm, corresponding to 99.5% demineralization.

[0151] Example 2 - Multi-column separation of tryptophan

[0152] In this example, the invention was carried out in chromatography mode on a continuous multi-column system for a fermentation slurry containing 40 g / L tryptophan at pH = 4. The main impurities were residues of the substrate, minerals and solid residues of cell metabolism.

[0153] The pH of the slurry was adjusted to 2.7 here by adding H2SO4. At this pH above the pKa, tryptophan is found mostly in the zwitterionic form.

[0154] Clarification is then carried out by tangential microfiltration on a ceramic membrane. Solid suspended material is retained by the membrane and concentrated in a fraction called the retentate. Tryptophan and most soluble impurities are transferred through the membrane towards a fraction called the permeate.

[0155] At the end of the concentration, the retentate can be diluted and then reconcentrated by adding water to the retentate compartment: this so-called diafiltration operation increases the overall tryptophan yield by reducing its residual amount in the retentate compartment.

[0156] The microfiltration permeate thus treated contained 98% of the tryptophan initially present in the slurry at a concentration of 15 g / L. The impurities here were mineral salts in an amount of 25 g / L: the value of their conductivity was 15.9 mS / cm. A sugar level of 3 g / L was also tested.

[0157] This permeate is used as the product for feeding the SSMB system described above.

[0158] According to the SSMB principle discussed previously, the system used in this specific case consists of 9 separation chambers used as columns and distributed as follows:

[0159] - Zone 1: three columns = elution zone of tryptophan;

[0160] - Region 2: two columns = separation region;

[0161] - Region 3: three bars = slowing region of tryptophan;

[0162] - Region 4: One column = loop region.

[0163] Each column contained 475 mL of XA2014-22 resin. The size of the bed volume is defined here as: 1 BV = 475 mL.

[0164] The initial state of the resin is that 3N concentration of ammonia NH3 is leaked to NH4 + form.

[0165] The system was operated at 4.5 BV / h.

[0166] The feed raw material product is injected at the top of zone 3. In each preparation cycle, 7.5 BV of the feed raw material product is injected. On the three columns, tryptophan is slowed down on the resin by the retardation phenomenon and is therefore separated from the impurities eluted and collected at the outlet of the zone in a fraction to be called "raffinate". In this raffinate, most of the impurities (salts and sugars) are eluted: its conductivity has a value of 17.6 mS / cm.

[0167] The system was adjusted so that 0.1% of the initial tryptophan was eluted in the raffinate fraction.

[0168] At the top of zone 1, ammonia diluted to 2 g / L (eluent) is injected, which desorbs tryptophan. In each preparation cycle, 5 BV of eluent is injected. During this elution, tryptophan is recovered in the so-called "extract" fraction. The conductivity of the extract is less than 200 μS / cm: the latter is demineralized in an amount of 98.8% relative to the feed raw product. A reduction of 89% in residual sugars and 75% in pigments was also determined.

[0169] A very small amount of ammonia eluent was used: the eluent / feed raw product ratio had a value of 0.67 (vol / vol). Furthermore, a concentration phenomenon was observed during this operation: tryptophan was eluted to 20 g / L (comparison: 15 g / L in the feed raw product). During this step, more than 99% of the initial tryptophan was recovered in the extract. In each SSMB cycle sequence, the amount of NH3 used was 0.59 mol / L resin (i.e., 0.4 mol NH3 / mol tryptophan) relative to a tryptophan injection of 1.47 mol / L resin: this proves that the retention mechanism is not ion exchange.

[0170] The curves tested in nine columns operated continuously within the loop step process are shown in Figure 3 middle.

[0171] Here, the distribution diagrams of tryptophan concentration, Brix (indicating the dry matter concentration) and conductivity (indicating the concentration of salt impurities) are depicted. Thus, the separation between the tryptophan elution zone (zone 1, columns 2, 3 and 4) and the raffinate collection zone (zone 3, columns 7, 8 and 9) is observed.

[0172] Example 3 - Retention study of phenylalanine

[0173] This example was performed with phenylalanine fermentation slurry, which was pretreated by adjustment to pH = 2 and then by microfiltration on a ceramic membrane.

[0174] Products XA2014-22 resin was used for treatment, 30 mL volume / column = 1 BV. The resin was converted into NH4 in advance by 3% NH3 + form, cleaned, and then balanced through a 2g / L NH3 pathway.

[0175] The chromatography feed material product included 32.76 g / L phenylalanine and had a conductivity of 36.8 mS / cm due to the salt concentration of the medium.

[0176] The feedstock product was injected in excess on the resin (4 BV / h): the retention of phenylalanine was observed relative to salts and sugars, followed by the leakage of the amino acid. Once the outlet concentration was equal to the inlet concentration of the amino acid, the injection of the feedstock product was stopped.

[0177] Elution of phenylalanine was achieved with NH3 at a concentration of 2 g / L at 4 BV / h.

[0178] Figure 4 The diagram shows the time-dependent variation of the concentration in the points of the system, where the entire separation step is seen to occur from the start of the raffinate elution to the end of the wash extract. Stage A is the injection stage of the initial mixture, and stage B is the elution stage with the NH3 solution.

[0179] The system provides the following operating possibilities:

[0180] - 169 g of phenylalanine per liter of resin were processed in a 6.75 hour production cycle.

[0181] - During the collection of the phenylalanine extract, the concentration was about 600 μS / cm, corresponding to a demineralization of 98.5%.

[0182] Example 4 - Retention study of histidine

[0183] This example was carried out with a 12.98 g / L histidine solution having a conductivity of 17.48 mS / cm and containing 3.9 g / L sugar. The solution was previously adjusted to pH = 2.7 by adding H2SO4.

[0184] The product XA2014-22 resin, 17 mL volume / column (= 1 BV). The resin was converted into NH4 in advance by 3% NH3. + form, washed, and then balanced at 2g / L through the NH3 pathway.

[0185] The feed raw material product was injected onto the resin in excess (2 BV / h): the retention of histidine was observed, followed by amino acid leakage. Once the output concentration was equal to the amino acid inlet concentration, the injection of the feed raw material product was stopped.

[0186] Elution of histidine was achieved with NH3 at a concentration of 2 g / L at 2 BV / h.

[0187] Figure 5 The diagram shows the time-dependent variation of the concentration in the points of the system, where the entire separation step is seen to occur from the start of the raffinate elution to the end of the wash extract. Stage A is the injection stage of the initial mixture, and stage B is the elution stage with the NH3 solution.

[0188] This system provides the following operating possibilities:

[0189] - 120 g of histidine per liter of resin were processed in a 30 hour production cycle.

[0190] - During the collection of the histidine extract, the concentration was about 220 μS / cm, corresponding to a demineralization of 98.8%.

Claims

1. A method for purifying amino acids from an initial mixture containing sugars and salts, wherein: The amino acid comprises an aromatic ring and is selected from phenylalanine or histidine, and has an acidity constant Ka, and the method comprises: - The first step: contacting the initial mixture with a strong cationic resin under the condition that the pH is greater than or equal to the pKa, - second step: eluting with an aqueous solution of an eluent having a pH value greater than that of the first step, thereby collecting a fluid rich in amino acids; wherein the second step of elution is performed directly after the first step of contacting without any intermediate washing, Wherein, the aqueous solution of the eluent is a solution of alkali in water, And, wherein the strong cationic resin is in the form of particles, and its average volume diameter (Dv50) is 20 to 350 μm, and wherein, in the amino acid-rich fluid, the total mass content of salt and the total mass content of sugar are reduced by at least 80% based on the total mass content of salt and the total mass content of sugar in the initial mixture, respectively; Wherein, the strong cationic resin is a polystyrene-divinylbenzene copolymer, and wherein the cross-linking rate of the strong cationic resin is 5 to 12%.

2. The method according to claim 1, wherein: The second elution step is an isocratic elution step.

3. The method according to claim 1 or 2, wherein: The amino acid has a basicity constant Kb, and during the first step, the pH value is less than or equal to pKb-3.

4. The method according to claim 1 or 2, wherein the alkali is soda, potash, ammonia or a mixture thereof.

5. The method according to claim 1 or 2, wherein: The concentration of the base in the aqueous solution of the eluent is 0.01 to 50 g / L.

6. The method according to claim 1 or 2, wherein: The ratio of the number of moles of the base in the aqueous solution of the eluent used in the second step to the number of moles of the amino acid in the initial mixture contacted in the first step is less than or equal to 3.

7. The method according to claim 1 or 2, wherein: The strong cationic resin is in the form of a monovalent cation.

8. The method according to claim 1 or 2, wherein: The method is carried out in a multi-column chromatography device.

9. The method according to claim 1 or 2, wherein: The method is carried out in a chromatography apparatus with a non-fixed bed.

10. The method according to claim 1 or 2, wherein: The initial mixture is obtained at the end of the treatment of the raw material mixture, and the treatment comprises one or more steps selected from centrifugation, pre-filtration, crystallization, tangential filtration, microfiltration and ultrafiltration.

11. The method according to claim 1 or 2, wherein: The method comprises one or more steps for concentrating the amino acid-rich fluid.

12. The method according to claim 11, wherein: At the end of the step for concentrating the amino acid-rich fluid, on the one hand the concentrated amino acid fluid is collected and on the other hand an eluent solution fluid is collected, the method comprising repeatedly circulating this eluent solution fluid towards the second elution step.

13. The method according to claim 1 or 2, wherein: The difference between the pH value of the aqueous solution of the eluent used in the second step and the pH value of the first step is 2 or more.

Images