SEPARATION AND PURIFICATION OF MONATIN

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims priority to U.S. Provisional Application Serial Number 61/335,033, filed 30 December 2009, entitled A POLYMER AND METHODS OF PREPARING AND USING A POLYMER; and U.S. Provisional Application Serial Number 61/335,037, filed 30 December 2009, entitled SEPARATION AND PURIFICATION OF MONATIN, which applications are incorporated herein by reference in their entirety.

FIELD

[0002] The present disclosure relates generally to a method and system for producing monatin. In particular, the present disclosure relates to a method and system for separating and purifying monatin from a mixture comprising monatin, starting materials and intermediates.

BACKGROUND

[0003] Monatin (2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid) is a naturally occurring, high intensity or high potency sweetener that was originally isolated from the plant Sclerochiton ilicifolius, found in the Transvaal Region of South Africa. Monatin has the chemical structure:

[0004] Because of various naming conventions, monatin is also known by a number of alternative chemical names, including: 2-hydroxy-2-(indol-3-ylmethyl)-4-aminoglutaric acid; 4-amino-2-hydroxy-2-(lH-indol-3-ylmethyl)-pentanedioic acid; 4-hydroxy-4-(3- indolylmethyl)glutamic acid; and 3-(l -amino- l ,3-dicarboxy-3-hydroxy-but-4-yl)indole.

[0005] Monatin has two chiral centers thus leading to four potential stereoisomeric configurations: the R,R configuration (the "R,R stereoisomer" or "R,R monatin"); the S,S configuration (the "S,S stereoisomer" or "S,S monatin"); the R,S configuration (the "R,S stereoisomer" or "R,S monatin"); and the S,R configuration (the "S,R stereoisomer" or "S,R monatin").

[0006] Reference is made to WO 2003/091396 A2, which discloses, inter alia, polypeptides, pathways, and microorganisms for in vivo and in vitro production of monatin. WO 2003/091396 A2 {see, e.g., Figures 1-3 and 11-13) and U.S. Patent Publication No. 2005/282260 describe the production of monatin from tryptophan through multi-step pathways involving biological conversions with polypeptides (proteins) or enzymes. One pathway described involves converting tryptophan to indole-3 -pyruvate ("1-3 -P") (reaction (1)), converting indole-3 -pyruvate to 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid (monatin precursor, "MP") (reaction (2)), and converting MP to monatin (reaction (3)). The three reactions can be performed biologically, for example, with enzymes.

SUMMARY

[0007] One embodiment is directed to a method of recovering monatin from a mixture including I3P, tryptophan, MP, and monatin, where the method includes the step of packing a chromatography column with a reverse phase resin comprised of particles, wherein the reverse phase resin has a particle size distribution in which about 90% of the particles are less than about 150 microns, pumping the mixture into the chromatography column, pumping the eluent through the chromatography column, removing a plurality of fractions from the column, wherein one or more of the fractions contains monatin having at least about 90% purity and collecting the one or more fractions containing monatin having at least about 90% purity. The method may further include the step of removing at least a portion of the tryptophan and the 13 P from the mixture, prior to pumping the mixture into the chromatography column. In one aspect, at least a portion of the tryptophan and the I3P from the mixture is performed by a nanofiltration membrane located upstream of the chromatography column.

[0008] Another embodiment is directed to a method of separating monatin from a mixture including monatin and one or more intermediates where the method includes the steps of packing a chromatography column with a resin, wherein the column includes a piston that contacts and exerts at least about 5 bar of pressure on the resin during operation of the chromatography column, pumping the mixture into the chromatography column using an elution pump, pumping an eluent through the chromatography column using the elution pump, removing a plurality of fractions from the chromatography column, wherein one or more of the fractions contains monatin having at least about 90% purity and opperating the chromatography column such that a back pressure on the elution pump is about equal to or greater than about 2 bar.

[0009] Yet another embodiment is directed to a method of removing monatin from a mixture comprising monatin and one or more intermediates, where the method includes the steps of adding the mixture to a chromatography column packed with a reverse phase resin, passing an eluent through the chromatography column, wherein the eluent is comprised essentially of water and eluting about 80% by weight of the monatin from the mixture in less than about 5 bed volumes, wherein the bed volume is equal to 1 liter of eluent per 1 liter of packed resin, and the eluted monatin has a purity of at least 90%. The method may further include the step of reducing a concentration of at least one of the one or more intermediates in the mixture using a nanofiltration membrane located upstream of the chromatography column. In one aspect, the nanofiltration membrane has a zeta potential of from about -19.0 to about -6.0.

[00 0] Still another embodiment is directed to a method of separating components from a mixture including monatin and tryptophan, where the method includes the steps of adding the mixture to a chromatography column packed with a reverse phase resin, passing an eluent through the chromatography column, wherein the eluent is comprised essentially of water, eluting monatin from the mixture and eluting at least about 10% by weight of the tryptophan from the mixture in less than about 30 bed volumes, wherein the bed volume is equal to 1 liter of eluent per 1 liter of packed resin.

[001 1 ] A further embodiment is directed to a method of recovering monatin from a mixture including I3P, tryptophan, MP, and monatin, where the method includes the steps of adding the mixture to a dynamic axial compression (DAC) chromatography column packed with a reverse phase resin, wherein a ratio of monatin to I3P in the mixture entering the chromatography column is less than about 1 , passing an eluent through the DAC chromatography column, wherein a plurality of fractions are removed from the column, and one or more of the fractions contains monatin having at least about 90% purity, and collecting the one or more fractions containing monatin having at least about 90% purity.

[0012] Another embodiment is directed to a method of recovering monatin from a mixture including I3P, tryptophan, MP and monatin, where the method includes the steps of passing a first mixture through a nanofiltration membrane to retain a second mixture having a lower concentration of I3P and tryptophan than in the first mixture, adding the second mixture to a dynamic axial compression (DAC) chromatography column packed with a reverse phase resin, wherein the second mixture has a ratio of monatin to BP greater than about 2, passing an eluent through the DAC chromatography column to elute monatin from the second mixture and collecting at least one fraction from the DAC chromatography column containing monatin having at least about 90% purity.

[0013] The details of one or more non-limiting embodiments of the invention are set forth in the description below. Other embodiments of the invention should be apparent to those of ordinary skill in the art after consideration of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is a block diagram of an exemplary system for the separation and purification of monatin from a mixture including monatin, starting materials and intermediates.

[0015] FIGS. 2A-2E are schematics illustrating operation of a dynamic axial compression (DAC) chromatography column for the separation and purification of monatin.

[0016] FIG. 3 is a schematic illustrating an example of the fractions removed from a DAC column as a function of bed volume.

[0017] FIG. 4 is a chromatogram showing elution of the various components, including monatin, from a chromatography column at bed volumes (L/L-R) between 0 and 20.

[0018] FIG. 5 shows a portion of the chromatogram from FIG. 4 at bed volumes (L/L- R) between 0 and 5.

[0019] FIG. 6 is a chromatogram showing elution of the various components from a chromatography column as a function of time.

[0020] FIG. 7 shows a portion of the chromatogram from FIG. 6 between 13 and 21 minutes, as monatin is eluted.

[0021 ] FIG. 8 is a chromatogram showing elution of the various components from a chromatography column as a function of bed volume (L/L-R) for an eluent containing 5% ethanol. [0022] FIG. 9 is a chromatogram showing elution of the various components from a chromatography column as a function of bed volume for an all-water eluent up to 7.5 BVs and a 10% ethanol eluent for the remaining 3 BVs.

[0023] FIG. 10 is a plot of peak resolution (Rs) between monatin precursor (MP) and monatin, and peak resolution (Rs) between monatin and indole-3-pyruvate (I3P), both as a function of the ethanol concentration of the eluent.

[0024] FIG. 11 is a plot of elution time for MP, monatin and I3P as a function of the ethanol concentration of the eluent.

[0025] FIG. 12A is a chromatogram showing elution of the various components from a chromatography column as a function of bed volume (L/L-R) for a resin having a larger bead size.

[0026] FIG. 12B shows a portion of the chromatogram from FIG. 12A at bed volumes between 0 and 8, and concentrations between 0 and 10 mmol/L.

[0027] FIG. 13A is a chromatogram showing elution of the various components from the chromatography column using the same resin as in FIG. 12 and operating the column at a higher temperature (about 60 degrees Celsius).

[0028] FIG. 13B shows a portion of the chromatogram from FIG. 13A at bed volumes between 0 and 7, and concentrations between 0 and 50 mmol/L.

[0029] FIG. 14 is a chromatogram showing elution of the various components from a chromatography column as a function of bed volume (L/L-R) in which an all-water eluent was used for about 40 BVs.

DETAILED DESCRIPTION

[0030] The present disclosure is directed to a method and system for separating and purifying monatin from a mixture including monatin, starting materials used in the production of monatin, and intermediates formed during the production of monatin. The method and system includes a chromatography unit that is packed with a reverse phase resin that is effective at separately eluting monatin from the other components in the mixture. The eluted monatin has a purity of at least 90%. In some embodiments, the eluted monatin is a stereoisomerically-enriched R,R monatin. In some embodiments, the chromatography unit is a dynamic axial compression (DAC) column. In some embodiments, the reverse phase resin is formed from polystyrene-divinylbenzene. [0031] Monatin has an excellent sweetness quality, and depending on a particular composition, monatin may be several hundred times sweeter than sucrose, and in some cases thousands of times sweeter than sucrose. As stated above, monatin has four stereoisomeric configurations. The S,S stereoisomer of monatin is about 50-200 times sweeter than sucrose by weight. The R,R stereoisomer of monatin is about 2000-2400 times sweeter than sucrose by weight. As used herein, unless otherwise indicated, the term "monatin" is used to refer to compositions including any combination of the four stereoisomers of monatin (or any of the salts thereof), including a single isomeric form.

[0032] Monatin may be synthesized in whole or in part by one or more of a biosynthetic pathway, chemically synthesized, or isolated from a natural source. If a biosynthetic pathway is used, it may be carried out in vitro or in vivo and may include one or more reactions such as the equilibrium reactions provided below as reactions (l)-(3). In one embodiment, is a biosynthetic production of monatin via enzymatic conversions starting from tryptophan and pyruvate and following the three equilibrium reactions below:

(1) Tryptophan Indole-3 -pyruvate (1-3-P)

+ Pyruvate ^{^} ^* ¹ + Alanine

Aminotransferase

(2) 1-3-P + Pyruvate ^{^} -* Monatin precursor (MP)*

Aldolase

(3) MP + Alanine ^ ^ Monatin + Pyruvate

Aminotransferase

* Monatin precursor (MP) is 2-hydroxy 2-(indol-3-ylmethyl)-4-keto glutaric acid.

[0033] The following side-reactions may also occur, resulting in production of hydroxymethyl-oxo-glutarate (HMO), hydroxymethylglutamate (HMG) or a combination thereof:

(4) Pyruvate + Pyruvate — ^{^ ' ^} HMO

Aldolase

(5) HMO + Alanine ^ ^{^} ^ ^{^} HMG + Pyruvate

D-Aminotransferase

[0034] In the pathway shown above, in reaction (1), tryptophan and pyruvate are enzymatically converted to indole-3 -pyruvate (1-3-P) and alanine in a reversible reaction. As exemplified above, an enzyme, here an aminotransferase, is used to facilitate (catalyze) this reaction. In reaction (1), tryptophan donates its amino group to pyruvate and becomes I-3-P. In reaction (1), the amino group acceptor is pyruvate, which then becomes alanine as a result of the action of the aminotransferase. The amino group acceptor for reaction (1) is pyruvate; the amino group donor for reaction (3) is alanine. The formation of indole-3-pyruvate in reaction (1) can also be performed by an enzyme that utilizes other a-keto acids as amino group acceptors, such as oxaloacetic acid and a-keto-glutaric acid. Similarly, the formation of monatin from MP (reaction (3)) can be performed by an enzyme that utilizes amino acids other than alanine as the amino group donor. These include, but are not limited to, aspartic acid, glutamic acid, and tryptophan.

[0035] Some of the enzymes useful in connection with reaction (1) may also be useful in connection with reaction (3). For example, aminotransferase may be useful for both reactions (1) and (3). The equilibrium for reaction (2), the aldolase-mediated reaction of indole-3 -pyruvate to form MP (i.e. the aldolase reaction), favors the cleavage reaction generating indole-3 -pyruvate and pyruvate rather than the addition reaction that produces the alpha-keto acid precursor to monatin (i.e. MP). The equilibrium constants of the amino transferase-mediated reactions of tryptophan to form indole-3 -pyruvate (reaction (1)) and of MP to form monatin (reaction (3)) are each thought to be approximately one. Methods may be used to drive reaction (3) from left to right and prevent or minimize the reverse reaction. For example, an increased concentration of alanine in the reaction mixture may help drive forward reaction (3). Reference is made to US Publication No. 2009/0198072 (Application Serial No. 12/315,685), which is also assigned to Cargill, the assignee of this application.

[0036] The overall production of monatin from tryptophan and pyruvate is referred to herein as a multi-step pathway or a multi-step equilibrium pathway. A multi-step pathway refers to a series of reactions that are linked to each other such that subsequent reactions utilize at least one product of an earlier reaction. In such a pathway, the substrate (for example, tryptophan) of the first reaction is converted into one or more products, and at least one of those products (for example, indole-3-pyruvate) can be utilized as a substrate for the second reaction. The three reactions above are equilibrium reactions such that the reactions are reversible. As used herein, a multi-step equilibrium pathway is a multi-step pathway in which at least one of the reactions in the pathway is an equilibrium or reversible reaction.

[0037] Because the R,R stereoisomer of monatin is the sweetest of the four stereoisomers, it may be preferable to selectively produce R,R monatin. For purposes of this disclosure, the focus is on the production of R,R monatin. However, it is recognized that the present disclosure is applicable to the production of any of the stereoisomeric forms of monatin (R,R; S,S; S,R; and R,S), alone or in combination.

[0038] In some embodiments, the monatin consists essentially of one stereoisomer - for example, consists essentially of S,S monatin or consists essentially of R,R monatin. In other embodiments, the monatin is predominately one stereoisomer - for example, predominately S,S monatin or predominately R,R monatin. "Predominantly" means that of the monatin stereoisomers present in the monatin, the monatin contains greater than 90% of a particular stereoisomer. In some embodiments, the monatin is substantially free of one stereoisomer - for example, substantially free of S,S monatin. "Substantially free" means that of the monatin stereoisomers present in the monatin, the monatin contains less than 2% of a particular stereoisomer. In some embodiments, the monatin is a stereoisomerically- enriched monatin mixture. "Stereoisomerically-enriched monatin mixture" means that the monatin contains more than one stereoisomer and at least 60% of the monatin stereoisomers in the mixture is a particular stereoisomer. In other embodiments, the monatin contains greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of a particular monatin stereoisomer. In another embodiment, a monatin composition comprises a stereoisomerically-enriched R,R-monatin, which means that the monatin comprises at least 60% R,R monatin. In other embodiments, stereoisomerically-enriched R,R-monatin comprises greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of R,R monatin.

[0039] For example, to produce R,R monatin using the three-step pathway shown above (reactions (l)-(3)), the starting material may be D-tryptophan, and the enzymes may be a D-aminotranferase and an R-specific aldolase. The three reactions, which are shown below, may be carried out in a single reactor or a multiple-reactor system.

(6) D-Tryptophan Indole-3 -pyruvate (1-3 -P)

+ Pyruvate + D-Alanine

D-Aminotransferase

(7) I-3-P + Pyruvate ^ ^ R-MP

R-specific Aldolase

(8) R-MP + D-Alanine ^ R,R-Monatin + Pyruvate

D-Aminotransferase [0040] In an embodiment in which a single reactor is used, the two enzymes (i.e. the D- aminotransferase and the R-specific aldolase) may be added at the same time and the three reactions may run simultaneously. The same enzyme may be used to catalyze reactions (6) and (8). A D-aminotransferase is an enzyme with aminotransferase activity that selectively produces, in the reactions shown above, D-alanine and R,R-monatin. An R-specific aldolase is an enzyme with aldolase activity that selectively produces R-MP, as shown in reaction (7) above. Although a focus in the present disclosure is on R,R monatin, it is recognized that the method and system of separating and purifying monatin is applicable to any of the stereoisomeric forms of monatin,.

[0041 ] There are multiple alternatives to the above pathway (i.e. reactions (6)-(8)) for producing R,R-monatin. For example, L-tryptophan may be used as a starting material instead of D-tryptophan. In that case, an L-aminotransferase may be used to produce indole- 3-pyruvate and L-alanine from L-tryptophan. Because L-alanine is produced, this pathway may require the use of an alanine racemase to convert the L-alanine to D-alanine, thus adding a fourth reaction to the monatin production pathway. (D-alanine is required to produce R,R monatin from the R- stereoisomer of monatin precursor (R-MP). In addition to requiring another enzyme (alanine racemase), undesired side reactions may also occur in this pathway. For example, L-alanine may react with the L-aminotransferase to produce R,S-monatin, or D-alanine may react with I-3-P to form D-tryptophan, resulting in a racemate of L-tryptophan and D-tryptophan, which has poor solubility. Some disadvantages of this pathway may be avoided by using a two reactor system as opposed to a single reactor system. It is recognized that there are additional alternatives not specifically disclosed herein for performing the three-step equilibrium pathway to produce monatin. The method and system described herein for separating and purifying monatin is applicable to monatin produced using alternative pathways to what is disclosed herein.

[0042] As described above, in some pathways, it may be preferable to perform the monatin producing reactions in two or more separate reactors, while in other pathways it may be preferable to use a single reactor system. The decision to use a one reactor or a multiple reactor system may depend, in part, on whether D-tryptophan or L-tryptophan is used as a starting material. A single reactor system is obviously simpler in design, eliminating the need for a second reactor, as well as eliminating, in some cases, a need for a separation step between the first and second reactors. It is recognized that the method and system described herein for separating and purifying monatin may be used in combination with both a single reactor system and a multiple reactor system for the production of monatin.

[0043] Although the present disclosure focuses on the production of monatin using the biosynthetic multi-step equilibrium pathway described above, monatin may also be produced chemically or using a combination of both chemical synthesis and an enzymatic pathway. Regardless of the method used to produce monatin, the resulting monatin may be present in a mixture that contains other components, including starting materials, intermediates, side products of the monatin-producing reactions or combinations thereof. It is preferable to separate the monatin from these other components, which may include, for example, tryptophan, pyruvate, alanine, I3P, MP, HMG and HMO. Thus, one aspect of the present disclosure is a method and system for separating and purifying monatin from this mixture using nanofiltration, chromatography or a combination thereof, such that the monatin has at least 90% purity.

[0044] After producing monatin, using either a single reactor or multiple reactor system, the monatin-containing mixture may undergo one or more intermediate processing steps before the chromatography process described herein. For example, the monatin- containing mixture may undergo a filtration process (also referred to herein as ultrafiltration or ultrafiltration process), upstream of the chromatography unit, which is designed to remove enzymes in the monatin-containing mixture. Another filtration process (referred to hereinafter as the nanofiltration process, nanofiltration, membrane filtration process or membrane process), also located upstream of the chromatography unit, may be used to remove some of the intermediates, such as BP and tryptophan, from the monatin-containing mixture, thus making the chromatography process more efficient. The nanofiltration process may also be used to concentrate permeate collected from the ultrafiltration process. In one aspect, enough water is removed to create a solution that is six times more concentrated than the original permeate from the ultrafiltration process. A concentration factor of six also helps compensate for the loss of permeate flux after the nanofiltration process stage. A further benefit of the nanofiltration process is that some of the other reaction components and intermediates are removed, allowing for stronger monatin peak resolution during operation of the chromatography unit. In one aspect, about 80% of pyruvate, about 30% tryptophan and almost about 50% of the BP and alanine are removed from the concentrate.

[0045] The purpose of the method and system described herein is to recover as close to 100%) as possible of the monatin produced, at a high purity level. It is recognized that although it may be possible to elute essentially all of the monatin from the chromatography column, if the monatin is not "pure" monatin, it is not defined herein as "recovered" monatin. As used herein, "pure" monatin is defined as a composition containing at least 90% by weight monatin, which is defined on a dry weight basis and corrected for inorganic counter ions. In some embodiments, the purity may be at least 95%; in other embodiments, at least 96%, at least 97%, at least 98%, and at least 99%.

[0046] Recovery is defined herein as the amount of pure monatin that is recovered from the mixture based on the starting mass of monatin. In some embodiments, about 80% by weight of the monatin, also on a dry weight basis, is recovered from the monatin- containing mixture. It is recognized that, in other embodiments, the system may be designed to recover less than 80% by weight of the monatin and/or recover monatin having a purity of less than 90%. It may be more efficient to recover less than 80%, depending, for example, on an overall system design for monatin production which includes recycle streams.

[0047] The separation and purification process described herein is designed, in large part, to maximize an overall carbon conversion efficiency within the monatin production system. As used herein "carbon" may refer to any of the starting materials, intermediates, or products in the monatin-producing pathway.

[0048] FIG. 1 is a block diagram of an exemplary system for the separation and purification of monatin. System 10 includes chromatography unit 12, feed inlet 14, eluent inlet 16, resin inlet 18, fraction outlet 20 and resin outlet 22. In some embodiments, chromatography unit 12 is located downstream of an enzyme removal unit. Chromatography unit 12 is a column packed with resin that forms a stationary phase in the chromatography separation process. The column is packed by loading resin into the column through resin inlet 18. At the end of operation, the resin may be removed from the column through resin outlet 22. The feed material (i.e. the monatin-containing mixture) is injected into the column through feed inlet 14. In one aspect, the monatin-containing mixture includes tryptophan, pyruvate, monatin, MP, I-3-P, alanine, HMO and HMG. The components in the monatin- containing mixture are adsorbed by the packed resin in the column. A mobile phase (an eluent) passes through the column through eluent inlet 16 and is designed to elute the components from the column through fraction outlet 20.

[0049] In some embodiments, the eluent is a mixture of water and an organic solvent that is miscible with water. These organic solvents may include but are not limited to acetonitrile, methanol or tetrahydrofuran, ethanol and 2-propanol (iso-propyl alcohol). The organic solvents may also include ethanol, isopropranol, methanol and mixtures thereof. In some embodiments, the eluent in system 10 is essentially all water, with no organic solvent.

[0050] A pump is used to inject the monatin-containing mixture into chromatography unit 12. The resin inside chromatography unit 12 causes the various components in the mixture to adsorb to the resin particles based on each component's affinity for the resin. The eluent is then pumped into chromatography unit 12 through eluent inlet 16. As the eluent passes through the column the components adsorbed by the resin in the column are eluted and flow out through the column with the eluent via fraction outlet 20. The most weakly adsorbed components (those with the lowest affinity for the resin) elute first. The most strongly adsorbed (i.e. highest affinity) elute last. The components may thus be separated by taking different fractions from the column. This may be done, for example, by transferring the outlet stream from fraction outlet 20 into a different container for each fraction. The fractionations from the column are described in further detail below in reference to FIG. 3. Depending on the volume of the monatin-containing mixture, multiple injections of the monatin-containing mixture through feed inlet 14 may be necessary.

[0051 ] In some embodiments, chromatography unit 12 uses reversed phase chromatography, meaning that the stationary phase or resin is non-polar. As compared to "normal" chromatography which uses a hydrophilic surface chemistry having a stronger affinity for polar compounds, in reversed phase chromatography the elution order of the components is reversed. The polar compounds are eluted first while non-polar compounds are retained. Thus the resin in reversed phase chromatography may be any inert non-polar substance; however, the particular composition of the resin directly impacts the separation behavior of the monatin-containing mixture, and small changes in the surface chemistries of the resin may lead to important changes in selectivity. When reversed phase chromatography is used to separate monatin from the other components in the monatin-containing mixture, the more polar compounds (such as, for example, pyruvate, alanine, HMO and HMG) are eluted first and are usually eluted closely together. The remaining components (such as, for example, MP, monatin, BP and tryptophan) elute more separately and in the following sequence - MP, monatin, I3P, and tryptophan.

[0052] The number of fractions taken from the column may depend in part on the selectivity of the resin to separate MP and monatin and to separate I3P and monatin. The number of fractions may also depend on whether any of the fractions are intended to be recycled back to other processing units with the monatin production system. In some embodiments, the first fraction from the chromatography column may include HMO, HMG, alanine and pyruvate. As stated above, these polar compounds usually elute closely together if a reversed phase resin is used in chromatography unit 12. In some embodiments, at least one fraction from the column contains monatin having a purity of at least 90%. In other embodiments, at least one fraction from the column contains monatin having a purity of at least 95%.

[0053] A relevant parameter in analyzing the performance of a resin for use in system 10 is the resin's ability to separate MP and monatin, and to separate monatin and BP. The separation between two components may be expressed as the peak resolution or "Rs", which is calculated using equation 9.

(9) RS = 2[AZ] / (W _A + WB),

where ΔΖ represents the separation between the two peaks, and W _A and W _B represent the widths at the base of peaks A and B. WA and WB are a function of the x-axis and therefore may be a variety of units such as time, volume, bed volume, etc. The Rs values for three different resins are shown in Table 4 below. Example 8 below compares peak resolutions between MP and monatin, and monatin and BP, for different eluent compositions.

[0054] The resin used in the chromatography process described herein is an adsorbent resin with a polystyrene-divinylbenzene matrix and without functionalized groups. In one embodiment, the resin has the properties shown in Table 1 below. As used herein, particle size values are diameters.

Table 1 - Resin Properties

[0055] In an alternative embodiment, the polymer described herein is an adsorbent resin with a polystyrene-divinylbenzene matrix and without functionalized groups having the properties listed in Table 2.

Table 2 - Resin Properties

[0056] It is recognized that reverse phase resins having properties outside the parameters shown in Tables 1 and 2 may be used in the method and system described herein for separation and purification of monatin. For example, in one aspect described herein is an adsorbent resin with a polystyrene-divinylbenzene matrix and without functionalized groups having properties listed in Table 3.

Table 3 - Resin Properties

[0057] In one embodiment, the resin has a particle size average of about 37 μηι, greater than 95%) of the particles have a diameter between about 30 and 50 μιη, the specific surface area is about 320 m ² /g, the specific pore volume is about 0.9 g/ml, and the average pore diameter is about 1 13 Angstrom. In another embodiment, the particle size average is between about 35 and 45 μπι, the specific surface area is about 626 m ² /g, the specific pore volume is about 1.4 g/ml, and the average pore diameter is about 88 Angstrom.

[0058] The monomers styrene and divinylbenzene may be used in various ratios to obtain a polystyrene divinylbenzene resin having the properties identified in Tables 1, 2 or 3. The amount of styrene in the resin may range from about 0.01 to 80% and the amount of divinylbenzene may range from about 20-99.99%). One of skill in the art would recognize that commercial sources of divinylbenzene may not be 100% pure and may contain other monomers. For example, in one aspect the commercially available divinylbenzene contains about 80% divinylbenzene and about 19%> other monomers where the other monomers include diethylenebenzene and styrene.

[0059] A resin having the properties in Tables 1, 2 or 3 and described herein may be made using the process described in U.S. Provisional Application Serial Number 61/335,033, entitled A POLYMER AND METHODS OF PREPARING AND USING A POLYMER.

[0060] Table 4 compares the separation performance of three different polystyrene- divinylbenzene resins. Resin batch #1 contains chloroform which is used as a porogen. Resin batches #2 and #3 are chloroform free.

Table 4: Comparison of three different resins

* Improvement in percent performance after optimization compared to original reverse phase resin with chloroform.

[0061 ] The results from Table 4 show that resin batch #3 (R102-1602) is significantly better than resin batch #1 (MH07-1234) in terms of faster elution of monatin (i.e. fewer bed volumes are required), faster elution of I3P and a lower degree of swelling. However, the peak resolution of MP and monatin for resin batch # 3 is lower than batches #1 or #2. Resin batch #3 requires less bed volume and thus less water required to elute monatin, as compared to both batches #1 and #2 (RH12-1517).

[0062] As shown in Table 1 above, the reverse phase resin may have a particle size distribution in which 96% of the resin particles are between about 50 and about 150 microns (μηι), less than 2% of the particles are greater than 150 microns, and less than 2% of the particles are less than 50 microns. In some embodiments, the reverse phase resin may have a particle size distribution in which 95% of the resin particles are between about 30 and about 50 microns. In some embodiments, the reverse phase resin has a particle size distribution in which about 90% of the particles are less than about 150 microns.

[0063] As mentioned above, the eluent in system 10 of FIG. 1 may be a mixture of water and an organic solvent (for example, ethanol), or the eluent may be essentially all water. A major advantage of using essentially all water as the eluent is simplification of operating system 10. When the eluent includes ethanol, an evaporation step is required downstream of chromatography unit 12 to remove the ethanol. This extra step increases production costs and requires additional environmental, health and safety precautions. As shown below in the examples, in those embodiments which include smaller resin particles, a similar separation of monatin is observed between an essentially all water eluent and an ethanol/water eluent.

[0064] In some embodiments, chromatography unit 12 utilizes dynamic axial compression (DAC) technology in combination with the resin described above. FIG. 2A-2E are schematics of a dynamic axial compression (DAC) chromatography column. An example of a DAC column that may be used is a ProChrom column from Novasep (Pompey, France). (

[0065] FIG. 2A is a schematic of DAC column 30 prior to loading of the resin into column 30. As shown in FIG. 2A, column 30 includes hydraulic jack 32, piston 34, column space 36, frit 38, and removable bottom plate 40. Piston 34 is a movable axially positioned piston, which is attached to hydraulic jack 32. Frit 38 is a filter or support configured to retain the resin inside column space 36, while allowing eluent to pass out of column space 36.

[0066] FIG. 2B is a schematic of DAC column 30 during loading of the resin into column space 36. Resin 42 is injected into column space 36 through resin slurry inlet 44. As shown in FIG. 2C, resin 42 is then packed down in space 36 by piston 34, creating resin bed 46. Column 30 is designed such that axial pressure by piston 34 results in a dynamic compression of resin 42. Thus piston 34 has a dynamic capability of maintaining a consistent bed compression by compensating for shrinking and/or swelling of resin 42. During operation of column 30, piston 34 constantly compensates for shifts in column bed stability and maintains resin bed 46 under a dynamic, adjustable compression.

[0067] FIG. 2D is a schematic of column 30 during operation. Piston 34 maintains pressure on resin bed 46. Through use of an eluent pump (not shown), eluent is pumped into column 30 through eluent inlet 48. Prior to pumping the eluent into column 30, the feed material (i.e. the monatin-containing mixture) is first pumped into column 30 via eluent inlet 48. Next, the eluent enters through eluent inlet 48 and the various components from the monatin-containing mixture are eluted in a particular order. As described above, different fractionations are taken from the column in order to have product streams with a particular composition. The fractions and the accompanying eluent exit column 30 through product outlet 50.

[0068] A volume of the feed material pumped into column 30 depends on the volume which may be adsorbed by resin bed 46. As such, multiple injections of the feed material, and consequently multiple passes of the eluent, may be required, depending on the volume of the monatin-containing mixture that is to be separated and purified.

[0069] As shown in FIG. 2D, eluent inlet 48 may be used as a product outlet, and product outlet 50 may be used as an eluent inlet. At the start of operating column 30, the feed material and the eluent are pumped through column 30 in a downward direction such that the elution exits through product outlet 50. In some embodiments, the direction of flow of the eluent is reversed, at some point during operation. The eluent is then pumped through column 30 in a upward direction such that the eluent enters column 30 through product outlet 50 and exits column 30 through eluent inlet 48.

[0070] Effective operation of column 30 depends, in part, on packing conditions for packing resin 42 in column space 36. As an example, the following packing conditions may be used for a DAC column having a diameter of 450 mm and intended to be packed to a height of about 500 mm. Prior to injecting resin 42 into column 30, resin 42 is slurried in a food grade azeotropic ethanol (96%) with a ratio of 3 kg ethanol per 1 kg resin. Resin 42 is agitated with an impeller agitator or equivalent in order to allow resin 42 to swell for at least about 3 hours. Resin 42 is transferred by gravity to column space 36 at a decreasing flow rate, ranging between about 5.2 L/h at the beginning to about 1.8 L/h at the end of the resin transfer. While resin 42 is in the ethanol phase, resin bed 46 is packed using piston 34 to ensure an adequate compression of bed 46. In some embodiments, piston pressure on resin 42 is about 5 bar. Once resin bed 46 is stable, bed 46 is eluted with water for about 15 minutes at a flow rate of about 5.7 L/h. Then bed 46 is further packed such that the piston pressure on resin 42 is between about 20 and about 30 bar.

[0071 ] A packing efficiency test may be performed on column 30. The following conditions are applicable to a column having a diameter of 450 mm and a packed bed height of about 500 mm. Fifty mL of a solution of sodium nitrate at 5 g/L (sodium nitrite may also be used) is injected into column 30 and then eluted with water (7 L/min). The absorbance of the liquor at outlet 50 is measured at 230 nm. Based on the position and geometry of the peak, the number of theoretical plates of the column can be calculated. In this example, the target value is greater than 1000 theoretical plates per meter.

[0072] As shown in FIG. 2D, piston 34 maintains pressure on resin bed 46 to maintain packing of resin 42 in column space 36. In some embodiments, the piston pressure during operation of DAC column 30 is at least about 5 bar; in other embodiments, the piston pressure is between about 10 bar and about 50 bar. In other embodiments, the piston pressure is about 20 bar; and in yet other embodiments, about 40 bar. Piston pressure may increase as a function of a decrease in the resin particle size.

[0073] As the eluent flows through column 30, a back pressure is created on the eluent pump. The back pressure is a function, in part, of the flow rate of the eluent and a length of column space 36. In some embodiments, the back pressure on the eluent pump is at least about 2 bar. For example, for a column having a bed height of about 500 mm, in some embodiments, the back pressure is at least about 3 bar; in other embodiments, the back pressure is between about 5 and about 10 bar. In other embodiments, the back pressure is between about 5 and about 40 bar, and in some embodiments, between about 30 and about 40 bar. A smaller particle size of the resin generally results in an increased back pressure on the eluent pump.

[0074] FIG. 2E is a schematic to illustrate unpacking of column 30 after operation of column 30 is complete. Bottom plate 40 and frit 38 are removed from column 30. Piston 34 is used to further press down on resin 42 and push resin 42 out of space 36.

[0075] As described above, in reversed phase chromatography, the less polar components (MP, monatin, 13 P and tryptophan) are eluted later. More specifically, tryptophan is the last component to be eluted due to its high affinity for the resin, and often tryptophan is eluted much later than its nearest component, I3P. As stated above, in some embodiments, the eluent is pumped through the column in two stages - with the first stage being a down flow and the second stage pumping the eluent in the opposite direction (up flow). This may be done, in part, to better elute the tryptophan. In other embodiments, instead of switching the direction of flow of the eluent, an all water eluent may be used in a first elution stage and a water/ethanol eluent may be used in the second stage in order to elute tryptophan.

[0076] In some embodiments using a DAC column in combination with a reverse phase resin, the following operating conditions may be used: An oxygen free environment is maintained due to instability of one or more intermediates (for example, I3P) in the presence of oxygen. As such, before starting the DAC column, the feed and elution tanks may be sparged with an inert gas such as nitrogen for example and then during operation, it may be kept under a nitrogen overlay. The temperature inside the DAC column may be maintained at an operating temperature between about 10 and about 30 degrees Celsius. In some embodiments, the temperature may be maintained at less than about 25 degrees Celsius. In other embodiments, the temperature is maintained between about 10 and about 18 degrees Celsius; in yet other embodiments, the temperature is maintained at about 15 degrees Celsius. The pH inside the DAC column prior to injection may be maintained between about 5.0 and about 9.0 depending, in part, on the pH and ionic strength of the eluent chosen.

[0077] FIG. 3 is a schematic illustrating an example of the fractionations that may be taken from DAC column 30 of FIG. 2. FIG. 3 shows the inlet and outlet streams from DAC column 30 as measured as a function of bed volume, and the components contained within each output stream or fractionation. Bed volume (BV) is defined as a ratio of the volume of eluent to the volume of the packed resin in the column. More specifically, bed volume (BV) as used herein is the number of liters of eluent per 1 liter of packed resin (L/L-R). Thus the bed volumes of elution increases as the total volume of eluent pumped through the column increases as a function of the operating time of the column. In the embodiment represented by FIG. 3, the resin packed in DAC column 30 is a polystyrene-divinylbenzene resin that has the properties listed in Table 1 above. In some embodiments, the resin packed in DAC column 30 is a polystyrene-divinylbenzene resin that has the properties listed in Tables 2 or 3 above. [0078] As shown in FIG. 3, a feed solution (i.e. the monatin-containing mixture) is pumped through the packed column in a down flow direction in order for the resin to adsorb the various components in the feed solution. The resulting initial fractionation taken off the column is a waste stream and the volume of the waste stream correlates to the volume of liquid displaced by the feed solution inside the column. Next, the eluent stream is pumped through the column in a downward or down flow direction for approximately 1.7 BVs; thus resulting in a total BV equal to 2.2. In the embodiment of FIG. 3, the eluent is essentially all water. Four fractions are eluted from the column between BVs of 0.5 and 2.2. Fraction 1 is removed after 0.7 BV (total BV equal to 1.2) and includes HMO, HMG, alanine, pyruvate (the polar compounds), and a low concentration of MP. Fraction 2 is removed after 0.15 BV (total BV equal to 1.35) and includes MP and monatin. Fraction 3 is removed after 0.45 BV (total BV equal to 1.8) and contains monatin having a purity of at least about 95%. Fraction 4 is removed after 0.30 BV (total BV equal to 2.1) and contains monatin and I3P. FIG. 3 illustrates that essentially all of the monatin is eluted in about 2 BVs.

[0079] Following fraction 4, a direction of flow of the eluent through column 30 is reversed such that the water eluent is passed through column 30 in an upward direction. The up flow of eluent is passed through the column for 6.9 BV. The remaining components adsorbed on the resin in the column are tryptophan and 13 P. The last fraction, fraction 5 is removed at a total BV equal to 9.0 and contains tryptophan and some 13 P.

[0080] The concentration of monatin contained in fractions 2 and 4 is much lower than the concentration of monatin in fraction 3. In some embodiments, fractions 1 , 2, 4 and 5 may be recycled back to various processing units within the monatin production system. For example, fractions 1 and 5 may be recycled back to the bioreactor where monatin is produced.

[0081 ] Referring back to FIG. 2, fractions 1 -4 exit column 30 through product outlet 50; fraction 5 exits column 30 through eluent inlet 48. Because the fractions are continuously eluted from product outlet 50, operation of column 30 includes a method of separately collecting each fraction. One of skill in the art would recognize that each of fractions 1-4 may be collected separately using a variety of methods.

[0082] The fractionation values disclosed above and shown in FIG. 3 are an example of where the fractionations may be taken in an exemplary embodiment. It is recognized that the fractions may be taken at different points depending, in part, on the desired purity and recovery of the monatin. In other embodiments, more than one fraction may contain a high purity of monatin, depending on the total number of fractions and the bed volume corresponding to each fraction. The number and timing of the fractions may also depend on whether any of the non-monatin components are being recycled back to the bioreactor or to other processing units within the monatin-producing system.

[0083] In some embodiments, about 80% by weight of the monatin from the monatin- containing mixture is eluted in less than about 5 BVs, and the eluted monatin has a purity of at least 90%. In other embodiments, about 80% by weight of the monatin is eluted in less than about 3 BVs. As stated above, tryptophan elutes much later than the other components in the monatin-containing mixture. (See, for example, FIG. 4 from Example 6. Also see FIG. 10 from Example 8.) In some embodiments, at least about 10% by weight of the tryptophan in the monatin-containing mixture is eluted in less than about 30 bed volumes. In other embodiments, at least about 90% by weight of the tryptophan was eluted in less than about 9 bed volumes. In some embodiments, the bed volume required to elute tryptophan is reduced by changing a direction of flow of the eluent from a down flow direction to an up flow direction.

[0084] As described above, intermediate processing units may be included in the monatin-producing system and located between the bioreactor and the chromatography unit. Depending on their existence and design, these other processing units may likely effect a composition of the input stream or feed material to the chromatography column. Without an intermediate processing step, there is a high level of impurities (for example, I3P) in the feed material injected into the chromatography column. In some embodiments, a ratio of monatin to I3P entering the column is less than about 1 ; in other embodiments, the ratio is less than about 0.5. Even at ratios of less than 0.5 (where there is a significant amount of I3P relative to monatin), the DAC column and the reverse phase resin disclosed herein make it feasible to recover monatin having at least 90% purity.

[0085] On the other hand, it is recognized that using another processing unit between the bioreactor and the chromatography column, to reduce the amount of one or more components of the monatin-containing mixture, including water, may increase the efficiency of the chromatography column. The increased efficiency may result in part from the fact that while the same amount of a particular component is present in a mixture, the overall volume of the mixture is reduced. In an embodiment, a DAC chromatography column is used in combination with a membrane located upstream of the chromatography column and configured to reduce the relative proportion of at least one or more of the intermediates, impurities or both in the mixture prior to adding the mixture to the chromatography column. Thus, in one aspect, the monatin purity of the column feed is increased prior to separation thereby increasing the efficiency of the chromatographic separation. In some embodiments, the membrane is a nano filtration membrane having a zeta potential of from about -19.0 to - 6.0 and monatin rejection values greater than 95%, greater than 96%, greater than 97% and in some instances greater than 98%. In an alternative embodiment, a reverse osmosis membrane may be used. A benefit of using a reverse osmosis membrane is that it is adapted to remove water and as such increase the concentration of the feed material.

[0086] Using a membrane upstream of the chromatography column, one or more of the intermediates from the monatin-containing mixture may be reduced. For example, the membrane may be used to reduce a concentration of 13 P and/or tryptophan in the mixture added to the chromatography column. In some embodiments, a mass ratio of monatin to I3P in the mixture entering the chromatography column is at least about 2; in other embodiments, at least about 3; and in yet other embodiments, at least about 5. Because the mixture entering the chromatography column contains less tryptophan relative to monatin, for example, in some embodiments, tryptophan is eluted from the mixture in less bed volumes, thus requiring less eluent and a faster elution time.

[0087] Aspects of the invention are illustrated in the following non-limiting examples.

EXAMPLES

Example 1

Derivatization of Monatin Intermediates (Indole-3 -Pyruvic Acid, Hydroxymethyloxyfilutaric Acid (HMO), Monatin Precursor, and Pyruvate) with Q-(4-Nitrobenzyl)hydroxylamine hydrochloride (NBHA)

[0088] In the process of monatin production various intermediate compounds are formed and utilized. These compounds include: indole-3 -pyruvic acid, hydroxymethyloxyglutaric acid (HMO), monatin precursor, and pyruvate. The ketone functional group on these compounds can be derivatized with 0-(4- Nitrobenzyl)hydroxylamine hydrochloride (NBHA) to form a stable compound for analysis.

UPLC/UV Analysis of Monatin Intermediates (Indole-3-Pyruvic Acid, Hydroxymethyloxyglutaric Acid, Monatin Precursor, and Pyruvate) [0089] A Waters Acquity UPLC instrument including a Waters Acquity Photo-Diode

Array (PDA) absorbance monitor is used for the analysis of the intermediate compounds. UPLC separations were made using a Waters Acquity HSS T3 1.8mm lxl 50mm column at 50° C. The UPLC mobile phase consisted of A) water containing 0.3% formic acid and 10 mM ammonium formate and B) 50/50 acetonitrile/methanol containing 0.3% formic acid and 10 mM ammonium formate.

[0090] The gradient elution was linear from 5% B to 40% B, 0-1.5 min, linear from 40% B, to 50% B, 1.5-4.5 min, linear from 50% B to 90% B, 4.5-7.5 min, linear from 90% B to 95%) B, 7.5-10.5 min, with a 3 min re-equilibration period between runs. The flow rate was 0.15 mL/min from 0-7.5 min, 0.18mL/min from 7.5-10.5min, 0.19mL/min from 10.5-1 1 min, and 0.15mL/min from 1 l -13.5min. PDA absorbance was monitored at 270nm.

[0091 ] Sample concentrations are calculated from a linear least squares calibration of peak area at 270nm to known concentration, with a minimum coefficient of determination of 99.9%.

Example 2

UPLC/UV Analysis of monatin and tryptophan

[0092] Analyses of mixtures for monatin and tryptophan derived from biochemical reactions were performed using a Waters Acquity UPLC instrument including a Waters Acquity Photo-Diode Array (PDA) absorbance monitor. UPLC separations were made using an Agilent XDB C8 1.8um 2.1x100mm column (part # 928700-906) at 30° C. The UPLC mobile phase consisted of A) water containing 0.1% formic B) acetonitrile containing 0.1 % formic acid.

[0093] The gradient elution was linear from 5% B to 40% B, 0-4 min, linear from 40% B, to 90% B, 4-4.2 min, isocratic from 90% B to 90% B, 4.2-5.2 min, linear from 90% B to 5% B, 5.2-5.3 min, with a 1.2 min re-equilibration period between runs. The flow rate was 0.5 mL/min, and PDA absorbance was monitored at 280nm.

[0094] Sample concentrations are calculated from a linear least squares calibration of peak area at 280nm to known concentration, with a minimum coefficient of determination of 99.9%. Example 3

Liquid Chromatography-Post Column Derivatization with OPA, Fluorescence Detection of Amino Acids, including: Hvdroxymethyl glutamate (HMG) and Alanine

[0095] Analyses of mixtures for HMG and alanine derived from biochemical reactions were performed using a Waters Alliance 2695 and a Waters 600 configured instrument with a Waters 2487 Dual Wavelengths Absorbance Detector and Waters 2475 Fluorescence Detector as a detection system. HPLC separations were made using both a Phenomenex Aqua C18 125A, 150mm x 2.1mm, 3μ, cat #00F431 1B0, and a Phenomenex Aqua C18 125A, 30mm x 2.1 mm, 3μ, cat # 00A431 1 B0 at 55° C. The HPLC mobile phase consisted of A) 0.6% acetic acid with 1 % MeOH.

[0096] The flow rate was (100% A) 0.2 mL/min from 0-3.5 min, 0.24 mL/min from 3.5-6.5min, 0.26 mL/min from 6.5-10.4 min, and 0.2 mL/min from 10.4-1 lmin. Absorbance was monitored at 336nm. Sample concentrations are calculated from a linear least squares calibration of peak area at 336nm to known concentration, with a minimum coefficient of determination of 99.9%.

Example 4

Chiral LC/MS/MS (MRM) Measurement of Monatin

[0097] Determination of the stereoisomer distribution of monatin in biochemical reactions was accomplished by derivatization with l -fluoro-2-4-dinitrophenyl-5-L-alanine amide 30 (FDAA), followed by reversed-phase LC/MS/MS MRM measurement.

LC/MS/MS Multiple Reaction Monitoring for the Determination of the Stereoisomer Distribution of Monatin

[0098] Analyses were performed using the Waters/Micromass® liquid chromatography-tandem mass spectrometry (LC/MS/MS) instrument including a Waters 2795 liquid chromatograph with a Waters 996 Photo-Diode Array (PDA) absorbance monitor placed in series between the chromatograph and a Micromass® Quattro Ultima® triple quadrupole mass spectrometer. The LC separations capable of separating all four stereoisomers of monatin (specifically FDAA-monatin) were performed on a Phenomenex Luna® 2.0 x 250mm (3 μιτι) CI 8 reversed phase chromatography column at 40°C. The LC mobile phase consisted of A) water containing 0.05% (mass/volume) ammonium acetate and B) Acetonitrile. The elution was isocratic at 13% B, 0-2 min, linear from 13%> B to 30% B, 2- 15 min, linear from 30% B to 80% B, 15-16 min, isocratic at 80% B 16-21 min, and linear from 80% B to 13% B, 21-22 min, with a 8 min re-equilibration period between runs. The flow rate was 0.23 niL/min, and PDA absorbance was monitored from 200 nm to 400 nm. All parameters of the ESI-MS were optimized and selected based on generation of deprotonated molecular ions ([M - H]-) of FDAA-monatin, and production of characteristic fragment ions. The following instrumental parameters were used for LC/MS analysis of monatin in the negative ion ESI/MS mode: Capillary: 3.0 kV; Cone: 40 V; Hex 1 : 15 V; Aperture: 0.1 V; Hex 2: 0.1 V; Source temperature: 120DC; Desolvation temperature: 350°C; Desolvation gas: 662 L/h; Cone gas: 42 L/h; Low mass resolution (Ql): 14.0; High mass resolution (Ql): 15.0; Ion energy: 0.5; Entrance: 0 V; Collision Energy: 20; Exit: 0 V; Low mass resolution (Q2): 15; High mass resolution (Q2): 14; Ion energy (Q2): 2.0; Multiplier: 650. Three FDAA- monatin-specific parent-to-daughter transitions were used to specifically detect FDAA- monatin. Identification of FDAA-monatin stereoisomers was based on chromatographic retention time as compared to purified monatin stereoisomers.

Example 5

Derivatization of Amino Acids with 9-fluorenylmethyl chloroformate (FMOC-chloride or

[0099] These amino acids include: Monatin, Alanine, Hydroxymethyl glutamate

(HMG), and Tryptophan. The amine functional group on these compounds can be derivatized with 9-fluorenylmethyl to form a stable compound for analysis.

UPLC UV Analysis of Monatin Amino Acids (Monatin, Alanine, Hydroxymethyl glutamate (HMG), and Tryptophan

[00100] A Waters Acquity UPLC instrument including a Waters Acquity Photo-Diode Array (PDA) absorbance monitor is used for the analysis of the intermediate compounds. UPLC separations were made using a Waters Acquity HSS T3, 100 mm x 2.1 mm x 1.8 μπι, (part #186003539) at 45° C. The UPLC mobile phase consisted of A) water containing 0.2% formic acid B) acetonitrile.

[00101] The gradient elution was linear from 10% B to 30% B, 0-1.0 min, linear from 30% B, to 37% B, 1.0-2.5 min, curved 7 from 37% B to 64% B, 2.5-5.7 min, curved 5 from 64% B to 90% B, 5.7-7.5 min, linear from 90% B to 95% B, 7.5-8.0 min, linear from 95% B to 10% B, 8.0-8.1 min, with a 1.4 min re-equilibration period between runs. The flow rate was 0.6 mL/min. PDA absorbance was monitored at 265nm. [00102] Sample concentrations are calculated from a linear least squares calibration of peak area at 265nm to known derivatized external standard, with a minimum coefficient of determination of 99.9%.

[00103] In Examples 6-13 below, the mass of each component in the inlet and/or outlet streams was determined using a combination of the analytical methods provided above in Examples 1-5.

Example 6

[00104] The present example evaluated the separation of the components in a monatin- containing mixture using a DAC column having a diameter of 30 cm. The column was operated at a piston pressure of about 20 bar. The resin packed in the column was a polystyrene-divinylbenzene cross-linked polymer in which 96% by volume of the resin particles were between about 50 and about 150 μιη and the average particle diameter by volume was 93 μπι.

[00105] The feed solution (i.e. the monatin-containing mixture) was first injected - 409 mL - at a flow rate of 800 mL/min. A water eluent was pumped downward through the column at a flow rate of 640 mL/min for a total volume of 16.5 L. A water eluent was then pumped upward (back flush) through the column at a flow rate of 4.7 L/min for a total volume of 135.9 L. The water eluent was degassed, deionized water with a conductivity of 1.67 uS. The temperature of the column and the eluent were between about 20 and about 25 degrees Celsius.

[00106] A 2 L fraction was taken from the column beginning at 2.05 BV (L/L-resin). The fraction contained 43% by weight of the monatin contained in the feed solution. The purity of the monatin in the fraction was 96% on a dry weight basis after correcting for salt, and essentially all of the monatin was R,R monatin (i.e. containing less than 1% of the other stereoisomer forms).

[00107] FIGS. 4 and 5 are chromatograms showing the separation profiles of the elutions from the DAC column. Monatin and seven other components - tryptophan, alanine, HMG, pyruvate, I3P, MP and HMO - each have a peak visible in the chromatogram. Two additional unidentified components are also visible in the chromatogram. FIGS. 4 and 5 confirm that the eight identified components are eluted in the order expected. As shown in FIG. 5, monatin is eluted after MP and before I3P. As such, any impurities in the fraction containing the monatin at 96% are likely to be MP and 13 P. Table 5 below shows the composition of the feed solution, as well as the composition of each of the fractions removed from the column.

Table 5: Composition of feed solution and fraction outlets

Example 7

[00108] The present example evaluated the separation of the components in a monatin- containing mixture using a DAC column having a diameter of 450 mm, a total length of 700 mm and a total volume of 1 1 1 L. The DAC column was operated at a piston pressure of at least 20 bar. The DAC column contained a HiPerSep module and is available from Novasep (Pompey, France). In this example, the feed solution (i.e. monatin containing mixture) had already passed through a nanofiltration (NF) membrane, specifically a GE DL membrane from GE. As such the feed solution to the DAC column contained a lower concentration of I3P as compared to the feed solution in Example 6. Further, the I3P concentration was higher but the ratio of monatin to 13 P was lower.

[00109] The resin packed in the DAC column was a polystyrene-divinylbenzene cross- linked polymer conforming to the characteristics described in Table 1 above. After packing and removal of fines, the volume of the resin in the column was 60.4 L, which corresponds with a bed height of 38 cm.

[001 10] The total volume of the feed solution was about 307 L, and at a density of 1.05 g L, the mass of the feed solution was about 322 kg. The concentration of monatin in the feed solution was 37.6 g/L; the total quantity of monatin in the feed solution was 1 1.54 kg. The concentration of D-tryptophan in the feed solution was 13.2 g/L.

[001 1 1 ] The volume of each feed injection was 3.19 L (5.3% BV) at a flow rate of 3.19 L/min, for a duration of about 1 minute. The quantity of monatin per injection was 120 g. The feed solution was injected in a downflow direction. The eluent was essentially all deoxygenated purified water. The first elution pass was in a downflow direction at a flowrate of 7 L/min or 7 BV/hour. The first elution ran for about 20 minutes. The second elution pass was in an upflow direction at a flow rate of 14 L/min or 14 BV/hour for about 39 minutes. Thus the duration of the run for each feed injection was about 1 hour. The total number of injections was 96, for a total operation time of about 96 hours. The operating temperature was at an ambient temperature ranging between about 15 and 25 degrees Celsius.

[001 12] The fractions were removed from the column between 0 and 60 minutes. FIG. 6 is a chromatogram showing the separation profile for one of the injections. Alanine, HMG, HMO, pyruvate and MP eluted between about 6 and about 14 minutes. Monatin eluted between about 14 and about 21 minutes. Tryptophan and BP eluted between about 21 and about 60 minutes. FIG. 7 is an enlarged portion of the chromatogram of FIG. 6 showing the monatin elution.

[001 13] For the monatin-containing fraction, the total volume collected was 3188 L, the total mass was 9707 grams, for a monatin concentration of 3.045 g/L. The monatin purity was 98.1 % (about 99% R,R-monatin) and the monatin recovery (on a dry weight basis) was 84.1 %.

Example 8

[001 14] In this example, a low pressure column (Ace Glass Cat. #5821-28) having a diameter of 2.5 cm and a height of 51 cm was packed with a polystyrene divinylbenzene cross-linked polymer having the properties shown in Table 1 above. The resin was wetted with 200-proof ethanol and loaded into the column and allowed to settle by gravity. A water eluent was pumped through the column at 12.5 mL/min or 3.0 BV/hr, and the resin compacted to a height of 51 cm. The feed solution (i.e. monatin containing mixture) - 53.98 grams - was pumped into the column. The column was first eluted with 0.8 BV (0.2 L) of water. Next the column was eluted with 10.75 L (4.3 BV) of a water eluent having 5% ethanol. The composition of the feed solution is shown in Table 6 below.

Table 6: Concentration of the components in the feed solution

[001 15] FIG. 8 is a chromatogram that illustrates the elution of each of the components in the feed solution as a function of bed volume (BV). All of the components had eluted within 6 BVs. More specifically, monatin eluted between about 1.7 and 2.5 BV, I3P eluted between about 2 and 3 BV, and tryptophan eluted between about 3.4 and 5.6 BV.

[001 16] Even though all of the monatin entering the column was eluted, about 51% of the recovered monatin was pure monatin (i.e. at least 90% of the total weight is monatin).

Example 9

[001 17] The present example evaluated the use of an all water eluent for about 7.5 BVs, as compared to the eluent in Example 8 above having 5% ethanol. In this example, the same column and resin were used as in Example 8.

[001 18] The resin was wetted with 200-proof ethanol, loaded into the column and allowed to settle by gravity. Next, the column was rinsed with deionized and deoxygenized (DI/DO) water for about at least 4 bed volumes before loading the feed solution. The feed solution (53.33 g), having the composition shown in Table 7 below, was pumped into the column. An all-water eluent was then pumped through the column in a down flow direction for about 7.5 BV (18.7 L). The column was then eluted for about 3.0 BV (0.75 L) with an eluent having 10% ethanol.

Table 7: Concentration of the components in the feed solution

[001 19] FIG. 9 is a chromatogram which illustrates the elution of the various components as a function of bed volume (BV). As shown in FIG. 9, monatin eluted between about 1.7 and 2.5 BV and tryptophan eluted between about 2 and 3.4 BV. This is similar to the elution time for monatin and tryptophan shown in FIG. 8. There wsa not an appreciable difference between the all-water eluent and 5% ethanol eluent, in terms of the elution time for the non-tryptophan components. However, the tryptophan eluted later in FIG. 9, as compared to FIG. 8. More specifically, 80% of the tryptophan eluted between about 6.0 and 9.1 BV for the water eluent. At 9.1 BVs, the eluent was switched to 10% ethanol, as which point the remaining 20% of the tryptophan was eluted.

[00120] In this example essentially none of the monatin recovered was pure monatin. Further, the data provided additional evidence that gravity packing may produce variable results. Packing in a DAC type column improved consistency.

Example 10

[00121 ] In the present example, further evaluation was conducted to compare the effectiveness, in terms of separation, of eluents having various ethanol concentrations. In this example, water eluents having ethanol concentrations of 0%, 1 %, 2%, 4%, 5% and 7% were tested in the low pressure column and using the same resin as in Examples 8 and 9 above.

[00122] FIG. 10 shows the peak resolution (Rs) between MP and monatin, as well as the peak resolution (Rs) between monatin and 13 P, for water eluents having ethanol concentrations of 0%, 1%, 2%, 4%, 5% and 7%. For ethanol concentrations of 0% and 2%, more than one data point was collected. A higher Rs value represents a better separation between the two respective components (i.e. monatin and MP; monatin and I3P). The results of FIG. 10 illustrate that there was no appreciable difference in separation as a function of the ethanol concentration.

[00123] FIG. 1 1 is a plot of elution time for MP, monatin, and I3P as a function of ethanol concentration. Consistent with FIG. 10, the results of FIG. 11 illustrate that there was no appreciable difference in elution time for MP, monatin, and 13 P at varying concentrations of ethanol.

Example 11

[00124] The present example further evaluates the effectiveness of a different resin for eluting monatin, 13 P and tryptophan. In contrast to Examples 8 and 9 above, the resin used in this example is a larger bead resin - SP850 from Mitsubishi Chemical Corporation. More specifically, for SP850, 90% of the particles are greater than 250 μηι, as compared to the resin in Examples 8 and 9 in which 90% of the particles are less than 150 μπι.

[00125] In this example a low pressure column (Ace Glass Cat # 5821 -28) having a diameter of 2.5 centimeters and a length of 54 centimeters was packed to a height of 54 centimeters with the SP850 resin. Water was rinsed from top to bottom through the column at 12.5 mL/min or 2.83 BV/hr. The temperature of the column was maintained at about 21 degrees Celsius during operation.

[00126] The feed solution (26.6 g), having the composition shown in Table 8 below, was introduced into the top of the column. The first elution was water, which was pumped through the column for 5 BVs (1.4 L), followed by 3 BVs (0.9 L) of a second elution of water having 50% ethanol.

Table 8: Concentration of the components in the feed solution

[00127] FIG. 12A is a chromatogram showing the separation profile up to 10 BVs. (After the 3 BVs of ethanol, approximately 2 BVs of water was eluted through the column to rinse the ethanol from the column.) As shown in FIG. 12A, the tryptophan was eluted in about 8 BVs due to the fact that the eluent was switched to an ethanol containing eluent at 5 BVs. All of the I3P and tryptophan were eluted during the second elution phase using water/ethanol. FIG. 12B shows a portion of the chromatogram of FIG. 12A up to a bed volume of 8 and at concentrations up to 10 mmol/L. Before changing to ethanol, about 71% by weight of the monatin (19.5 mg) was eluted. The remaining monatin - about 29% (8.0 mg) - was eluted using the water/ethanol elution. The amount of monatin recovered was about 29% at greater than 90% purity. Example 12

[00128] In the present example, the same column and resin were used as in Example 1 1. The resin was packed to a height of 54 centimeters and water was rinsed from top to bottom through the column at 12.5 mL/min or 2.83 BV/hr. The difference in this example was that the column was maintained at a temperature of about 60 degrees Celsius. The feed solution (125.89 g) was introduced into the top of the column. The composition of the feed solution is shown in Table 9 below.

Table 9: Concentration of the components in the feed solution

[00129] The first elution of water was pumped through the column for 7 BV (1.8 L) at a flow rate of 12.5 mL/min. The second elution of water and 50% ethanol was pumped through the column for 3 BV. FIG. 13 A is a chromatogram which shows the separation of the various components. FIG. 13B shows a portion of the chromatogram of FIG. 13A up to a bed volume of 7 and at concentrations up to 50 mmol/L. Essentially all of the monatin was eluted in 7 BVs; about 99% of the I3P eluted in 7 BVs. The tryptophan was not eluted until the eluent was switched to the water/ethanol mixture.

[00130] In this example, a membrane pretreatment step was utilized. This resulted in a higher concentration of monatin relative to I3P and tryptophan. The amount of monatin recovered was about 12% at greater than 90% purity.

Example 13

[00131 ] In the present example the same column and resin were used; the water eluent was run for a longer period of time to determine if and when tryptophan would elute using an all water eluent. The SP850 resin was loaded into the column to a height of 54 centimeters. Water was rinsed from top to bottom through the column at 12.5 mL/min or 2.83 BV/hr. The feed solution (28.45 g) was introduced through the top of the column. The composition of the feed solution is shown in Table 10 below.

Table 10: Concentration of the components in the feed solution

[00132] The all water eluent was pumped through the column for about 40 BVs (10.6 L). A 15% ethanol eluent was then pumped through the column for 12.25 L.

[00133] FIG. 14 is a chromatogram showing the separation of the various components as a function of bed volume. As shown in FIG. 14, after pumping an all water eluent through the column for 40 bed volumes, about 5% of the tryptophan had eluted. About 95% of the tryptophan eluted once the 15% ethanol eluent was used.

[00134] The amount of monatin recovered was 0% at greater than 90% purity.lt is recognized that various modifications to the described invention may be made without departing from the spirit and scope of the disclosure. It is recognized that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Example 14

[00135] In this example, a DAC column having a diameter of 1 1 cm was packed with a polystyrene-divinylbenzene cross-linked polymer resin in which at least 95%> of the beads were of a diameter between 30 μηι and 50 μπι, having an average diameter or particle size of 37 μηι. The porosity characteristics of the resin were as follows: surface area equal to 320 m ² /g, total pore volume equal to 0.9 g/ml, and average pore diameter equal to 1 13 Angstrom. The resin was packed in an Ethanol slurry (200 proof) of about 0.75 kg resin to about 2.6 kg Ethanol. The piston pressure was set to about 10 bar to expel excess solvent. Water was passed through the column at a flow rate of 300 mL/min until 4 bed volumes had passed, at which point the piston pressure was increased to about 40 bar and maintained at that pressure during operation. The bed height of the packed resin was 22 cm.

[00136] The feed solution - 8.82 kilograms (containing approximately 533 grams of monatin) - was passed over the column in numerous injection elution cycles. Approximately 57 injections were conducted with an injection size of approximately 145 mL. Following each injection into the top of the column, water was passed from the top down at a rate of 600 mL/min until the monatin was fully eluted, at which point the flow rate was increased to 800 mL/min and the direction was reversed. Degassed, deionized water was used as the eluent in all steps.

[00137] A total of six (6) fractions were collected during each chromatography cycle. An initial waste fraction of primarily water only was collected for the first 0.72 bed volume (BV) from each cycle. Fraction 1 was collected for the next 0.5 BV in each cycle. Fraction 2 was collected for the next 0.08 BV in each cycle. Fraction 3 was collected for the next 0.33 BV in each cycle. Fraction 4 was collected for the next 0.18 BV in each cycle. Backflush was initiated at the higher flowrate, and Fraction 5 was collected for the next 8.97 BV in each cycle. The composition of each fraction is shown in Table 1 1 below.

Table 11: Concentration of the components in the feed solution and fractions

*There are three species of monatin precursor structural isomers (MPSI) and four species of BP dimer. [00138] Fractions 1 and 5 contain mostly non-monatin components. Fractions 2 and 4 contain monatin, but with other components at levels higher than desired for a finished product. Fraction 3 contains monatin at purity greater than 90%. The back pressure during monatin elution was approximately 33 bar.

Example 15

[00139] The monatin and other components in Table 11 of Example 14 were measured using the following analytical methods:

Pyruvate and HMO Determination by UHPLC/UV

[00140] Analyses of mixtures derived from biochemical reactions or purge samples from the purification process were performed using UHPLC separations after derivatization with o-phenylenediamine (OPD) on a Waters Acquity UPLC instrument including a Waters Acquity Photo-Diode Array (PDA) absorbance detector. UHPLC separations were made using a Waters Acquity HSS T3 1.8um 2.1x30mm column at 30 oC. The UHPLC mobile phase consisted of A) water containing 0.2% formic acid and B) acetonitrile.

[00141] The gradient elution was linear from 13% B to 15% B, 0-1 min, linear from 15% B to 90% B, 1-2 min, isocratic at 90% B, 2.0-2.2 min, linear from 90% B to 13% B, 2.2- 2.3 min, with a 0.7 min re-equilibration period between runs. The flow rate was 0.6 mL/min and PDA absorbance was monitored at 338 nm.

[00142] Pyruvate and HMO concentrations are calculated from peak area as compared to an external standard of the same species.

Alanine and HMG Determination by UHPLC/UV

[00 43] Analyses of mixtures derived from biochemical reactions or purge samples from the purification process were performed using UHPLC separations after derivatization with 9-fiuorenylmethyl chloroformate (FMOC-C1) on a Waters Acquity UPLC instrument including a Waters Acquity Photo-Diode Array (PDA) absorbance detector. UHPLC separations were made using a Waters Acquity HSS T3 1.8um 2.1x30mm column at 30 oC. The UHPLC mobile phase consisted of A) water containing 0.2% formic acid and B) acetonitrile.

[00144] The gradient elution was linear from 25% B to 35% B, 0-1 min, linear from 35% B to 40% B, 1-2.5 min, linear from 40% B to 90% B, 2.5-3.0 min, isocratic at 90% B, 3.0-3.7 min, linear from 90% B to 25% B, 3.7-3.8 min, with a 0.7 min re-equilibration period between runs. The flow rate was 0.6 mL/min and PDA absorbance was monitored at 265 nm.

[00145] Alanine and HMG concentrations are calculated from peak area as compared to an external standard of alanine.

Total Indole Determination by UHPLC/UV

[00146] The remaining components shown in Table 1 1 , including monatin, were measured using the following method.

[00147] Analyses of mixtures derived from biochemical reactions or purge samples from the purification process were performed using UHPLC separations on a Waters Acquity UPLC instrument including a Waters Acquity Photo-Diode Array (PDA) absorbance detector. UHPLC separations were made using a Waters Acquity HSS T3 1.8um 2.1x100mm column at 45 oC. The UHPLC mobile phase consisted of A) water containing 10 mM ammonium formate and 0.1% formic acid and B) acetonitrile.

[00148] The gradient elution was isocratic at 5% B, 0-1.5 min, linear from 5% B to 12% B, 1.5-3.5 min, linear from 12% B to 35% B, 3.5-9.0 min, linear from 35% B to 95% B, 9.0- l l .O min, isocratic at 95% B, 11.0-12.0 min, linear from 95% B to 5% B, 12.0-12.1 min, with a 2.9 min re-equilibration period between runs. The flow rate was 0.6 mL/min and PDA absorbance was monitored at 280 nm.

[00149] Monatin and other non-monatin compound concentrations are calculated from the peak area as compared to an external standard of R,R-monatin with the following exceptions: concentrations of tryptophan and indole-3 -pyruvate (I3P) are calculated from the peak area as compared to an external standard of tryptophan and I3P, respectfully, while concentrations of monatin precursor (MP) and MP lactols are determined from the peak area as compared to an external standard of MP.

[00150] It is recognized that various modifications to the described invention may be made without departing from the spirit and scope of the disclosure. It is recognized that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXEMPLARY EMBODIMENTS

A. A method of recovering monatin from a mixture including monatin and at least one of BP, tryptophan, and MP, the method comprising:

packing a chromatography column with a reverse phase resin comprised of particles, wherein the reverse phase resin has a particle size distribution in which about 90% of the particles are less than about 150 microns;

pumping the mixture into the chromatography column;

pumping the eluent through the chromatography column;

removing a plurality of fractions from the column, wherein one or more of the fractions contains monatin having at least about 90% purity; and collecting the one or more fractions containing monatin having at least about 90% purity.

B. The method of claim A wherein the chromatography column includes an elution pump for pumping the mixture and the eluent through the column, and a back pressure on the elution pump is about equal to or greater than about 2 bar.

C. The method of claim A wherein the mixture includes monatin and BP and wherein a ratio of monatin to BP in the mixture entering the chromatography column is equal to or less than about 1.

D. The method of claim A wherein at least about 40% by weight of the monatin in the mixture is recovered in the one or more fractions containing monatin having at least about 90% purity.

E. The method of claim A further comprising:

removing at least a portion of the tryptophan and the BP from the mixture, prior to pumping the mixture into the chromatography column, wherein the mixture includes monatin, BP and tryptophan.

F. The method of claim E wherein removing at least a portion of the tryptophan and the BP from the mixture is performed by a nano filtration membrane located upstream of the chromatography column.

G. The method of claim F wherein the nanofiltration membrane has a zeta potential of about -19 to about -6.

H. The method of claim E wherein at least about 80% by weight of the monatin in the mixture is recovered in the one or more fractions containing monatin having at least about 90% purity. I. The method of claim A wherein the chromatography column is a dynamic axial compression (DAC) chromatography column.

J. The method of claim A further comprising:

operating the chromatography column such that a temperature of the mixture in the column is less than about 25 degrees Celsius.

K. The method of claim A wherein one or more of the fractions removed from the column contains monatin having at least about 95% purity.

L. The method of claim A wherein the monatin is a stereoisomerically-enriched R,R monatin.

M. The method of claim A wherein the reverse phase resin is a polystyrene- divenylbenzene resin.

N. A method of separating monatin from a mixture including monatin and one or more intermediates, the method comprising:

packing a chromatography column with a resin, wherein the column includes a piston that contacts and exerts at least about 5 bar of pressure on the resin during operation of the chromatography column;

pumping the mixture into the chromatography column using an elution pump;

pumping an eluent through the chromatography column using the elution pump;

removing a plurality of fractions from the chromatography column, wherein one or more of the fractions contains monatin having at least about 90% purity; and operating the chromatography column such that a back pressure on the elution pump is about equal to or greater than about 2 bar.

O. The method of claim N wherein the back pressure on the elution pump is between about 5 bar and about 10 bar.

P. The method of claim N wherein the piston exerts between about 10 bar and about 30 bar of pressure on the resin during operation of the chromatography column.

Q. The method of claim N wherein the eluent is water.

R. The method of claim N wherein the chromatography column is a dynamic axial compression (DAC) chromatography column.

S. The method of claim N wherein the resin is polystyrene-divenylbenzene.

T. The method of claim N wherein the resin is comprised of particles and has a particle size distribution in which 90% of the particles are less than 150 microns.

U. A method of removing monatin from a mixture comprising monatin and one or more intermediates, the method comprising: adding the mixture to a chromatography column packed with a reverse phase resin; passing an eluent through the chromatography column, wherein the eluent is comprised essentially of water; and

eluting about 80% by weight of the monatin from the mixture in less than about 5 bed volumes, wherein the bed volume is equal to 1 liter of eluent per 1 liter of packed resin, and the eluted monatin has a purity of at least 90%. V. The method of claim U further comprising:

reducing a concentration of at least one of the one or more intermediates in the mixture, prior to adding the mixture to the chromatography column.

W. The method of claim V wherein the one or more intermediates includes tryptophan. X. The method of claim V wherein the one or more intermediates includes BP.

Y. The method of claim V wherein reducing a concentration of at least one of the one or more intermediates in the mixture is performed by a nanofiltration membrane located upstream of the chromatography column.

Z. The method of claim Y wherein the nanofiltration membrane has a zeta potential of about -19 to about -6.

AA. The method of claim V wherein about 80% by weight of the monatin is eluted from the mixture in less than about 3 bed volumes.

BB. The method of claim U wherein the mixture entering the chromatography column comprises tryptophan and the method further comprises:

eluting about 90%> by weight of the tryptophan from the mixture in less than about 9 bed volumes.

CC. The method of claim U further comprising:

reversing a direction of flow of the eluent through the chromatography column.

DD. The method of claim U wherein the reverse phase resin is a polystyrene- divenylbenzene resin.

EE. The method of claim U wherein the reverse phase resin is comprised of particles and has a particle size distribution in which 90% of the particles are less than 150 microns.

FF. The method of claim U wherein the chromatography column is a dynamic axial compression (DAC) chromatography column.

GG. The method of claim U further comprising:

operating the chromatography column such that a temperature of the eluent and mixture inside the column is less than about 25 degrees. HH. The method of claim GG wherein the temperature is between about 10 and about 18 degrees.

II. A method of separating components from a mixture including monatin and tryptophan, the method comprising:

adding the mixture to a chromatography column packed with a reverse phase resin; passing an eluent through the chromatography column, wherein the eluent is comprised essentially of water; and

eluting the monatin from the mixture; and

eluting at least about 10% by weight of the tryptophan from the mixture in less than about 30 bed volumes, wherein the bed volume is equal to 1 liter of eluent per 1 liter of packed resin.

JJ. The method of claim II wherein about 80% by weight of the monatin is eluted from the mixture in less than about 5 bed volumes.

KK. The method of claim JJ wherein the eluted monatin has a purity of at least 90%.

LL. The method of claim II wherein about 90% by weight of the tryptophan is eluted in about 9 bed volumes.

MM. The method of claim II wherein the reverse phase resin is a polystyrene- divenylbenzene resin.

NN. The method of claim II wherein the reverse phase resin is comprised of particles and has a particle size distribution in which 90% of the particles are less than 150 microns.

OO. The method of claim II further comprising:

reversing a direction of flow of the eluent through the chromatography column prior to eluting the tryptophan from the mixture.

PP. The method of claim II further comprising:

operating the chromatography column such that a temperature of the eluent and the mixture inside the column is less than 25 degrees Celsius.

QQ. The method of claim PP wherein the temperature is less than about 18 degrees Celsius.

RR. A method of recovering monatin from a mixture including BP, tryptophan, MP, and monatin, the method comprising:

adding the mixture to a dynamic axial compression (DAC) chromatography column packed with a reverse phase resin, wherein a ratio of monatin to BP in the mixture entering the chromatography column is less than about 1 ; passing an eluent through the DAC chromatography column, wherein a plurality of fractions are removed from the column, and one or more of the fractions contains monatin having at least about 90% purity; and

collecting the one or more fractions containing monatin having at least about 90% purity.

SS. The method of claim RR wherein the ratio of monatin to 13 P in the mixture entering the chromatography column is about equal to or less than about 0.5.

TT. The method of claim RR wherein the reverse phase resin is comprised of particles and has a particle size distribution in which 90% of the particles are less than 150 microns.

UU. The method of claim RR wherein one or more of the fractions removed from the column contains monatin having at least about 95% purity.

VV. A method of recovering monatin from a mixture including I3P, tryptophan, MP and monatin, the method comprising:

passing a first mixture through a nanofiltration membrane to retain a second mixture having a lower concentration of I3P and tryptophan than in the first mixture; adding the second mixture to a dynamic axial compression (DAC) chromatography column packed with a reverse phase resin, wherein the second mixture has a ratio of monatin to I3P greater than about 2;

passing an eluent through the DAC chromatography column to elute monatin from the second mixture; and

collecting at least one fraction from the DAC chromatography column containing monatin having at least about 90% purity.

WW. The method of claim VV wherein at least about 80% by weight of the monatin in the second mixture is recovered in the at least one fraction from the DAC chromatography column.

XX. The method of claim VV wherein the second mixture has a ratio of monatin to 13 P greater than about 3.

YY. The method of claim VV wherein the second mixture has a ratio of monatin to I3P greater than about 5.