FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules  2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. METHODS FOR THE PRODUCTION OF L-CARNITINE

2.

1. (A) LONZA Ltd
(B) Switzerland
(C) Münchensteinerstrasse 38 CH-4002 Basel Switzerland

The following specification particularly describes the invention and the manner in which it is to be performed.

Methods for the production of L-carnitine

The invention relates to methods for the production of L-carnitine.

Background of the invention

Carnitine (vitamin Bt; 3-hydroxy-4-trimethylammonio-butanoate) is a quaternary ammonium compound biosynthesized from the amino acids lysine and methionine. In living cells  it is required for the transport of fatty acids from the cytosol into the mitochondria during the breakdown of lipids for the generation of metabolic energy. It is used as a nutritional supplement.

Carnitine exists in two stereoisomers. The biologically active form is L-carnitine  whilst its enantiomer  D-carnitine  is biologically inactive. When producing L-carnitine in an industrial process  it is desirable to produce the biologically active L-form in high purity. Highly pure L-carnitine can be obtained by microbiological processes. EP 0195944 discloses a microbiological process  in which L-carnitine is produced from crotonobetain and butyrobetain in a bioreactor with the aid of specific microorganisms. A mixture of enantiomeric pure L-carnitine with butyrobetain is obtained. In order to remove the butyrobetain  the final product is recrystallized with methanol and isobutanol.

Another microbiological process for obtaining essentially pure L-carnitine is disclosed by DD 296702. The culturing solution is depleted of L-carnitine by electrodialysis and recrystallization of the L-carnitine. In such microbiological processes  essentially no D-carnitine is produced and thus an enantiomeric separation step is not necessary. A recrystallization of L-carnitine from solvents is applied for removing other substances.

Obtaining highly pure L-carnitine is more complicated when using a non-microbiological or non-enzymatic process. By means of organic synthesis  usually a mixture of D- and L-carnitine is obtained. In order to obtain pure L-carnitine  DE 689 01 889 T2 suggests to apply a ruthenium phosphine complex in a stereoselective asymmetric hydrogenization. Such complexes are relatively complicated and expensive and thus not applicable for the preparation of large amounts of L-carnitine in an industrial process.

Thus methods have been developed for isolating L-carnitine from a mixture of L- and D-carnitine. In general  the methods are based on the conversion of carnitine into a salt with an optically active acid and separation of L- and D-carnitine due to different physical properties  such as solubility.

In this respect  DD 93 347 discloses a method for separating D- and L-carnitine in the presence of camphoric acid  dibenzoyl tartric acid or combinations thereof from alcoholic solutions. The D-carnitine is separated from the L-carnitine due to the different solubility of the salts.

DE 35 36 093 discloses a method for the preparation of L-carnitine from a racemic mixture  in which the D- and L-carnitine are converted to optically active salts with dibenzoyl-L-tartric acid followed by a fractionated crystallization.

However  methods in which the carnitine is converted into an optically active salt or acid are relatively complicated  because they comprise the steps of adding a separating agent and removing it after the separation process. This renders the overall process relatively time- and labour-efficient.

Problem underlying the invention

The problem underlying the invention is to provide a process for the production of L-carnitine  which overcomes the above-mentioned problems.

The process should be applicable for producing highly pure L-carnitine from an enantiomeric mixture comprising L- and D-carnitine. The enantiomeric purity shall be increased significantly thereby and the yield shall be high.

The method shall be carried out in a simple manner. Specifically  the use of additional compounds  such as optically active auxiliary agents  which have to be removed afterwards  shall be avoided. Further  the number of process steps shall be relatively low and the process shall not require complicated apparatuses. Overall  the process shall be cost- and labour-efficient.

Disclosure of the invention

Surprisingly  the problem underlying the invention is solved by the method according to the claims. Further inventive embodiments are disclosed throughout the description.
Subject of the invention is a method for the production of L-carnitine  comprising the steps of
(a) providing a solution comprising at least 5% (w/w) carnitine in a first solvent  wherein the carnitine is a mixture of D- and L-carnitine 
(b) optionally seeding the solution with L-carnitine crystals 
(c) adding a second solvent  in which the L-carnitine is not soluble or has a low solubility 
(d) isolating crystals comprising L-carnitine.

Carnitine is a zwitterion  which comprises a carboxy group and a quarternary ammonium group. The carnitine used in step (a) is preferably this zwitterionic carnitine. However  it is also possible to use a salt  such as a chloride  sulphate or nitrate salt. The carnitine used in step (a) is preferably not a salt of a carnitine with an optically active anion.

By the method of the invention  L-carnitine is obtained at enhanced purity. The method of the invention is thus also a method for purifying L-carnitine  or a method for obtaining a highly pure L-carnitine  or a method for increasing the enantiomeric excess of L-carnitine.

According to the invention  crystalline L-carnitine is obtained from a solution comprising D- and L-carnitine. In a preferred embodiment  the solution in step (a) essentially consists of the solvent and carnitine. In the method of the invention  the enantiomeric excess (e.e.) of L-carnitine is enhanced. Preferably  the enantiomeric excess is enhanced by more than 1% or more than 2%. The increase of the enantiomeric excess depends on the enantiomeric excess in the initial solution in step (a). The enantiomeric excess (e.e.) is defined as the absolute difference between the mole fractions of each enantiomer in percent. As an example  a sample with 90% of L-isomer and 10% of D-isomer has an enantiomeric excess of 80% L-isomer.

In preferred embodiments of the invention  in step (a) the carnitine comprises more than 50%  80%  90%  95% or 98% (e.e.) L-carnitine. Preferably  the L-carnitine isolated in step (d) comprises more than 90%  98%  99% or 99.5% (e.e.) L-carnitine. Preferably  when a solution in step (a) comprising more than 95% (e.e.) L-carnitine is used  in step (d) crystals comprising more than 99% (e.e.) L-carnitine are obtained.

In a preferred embodiment of the invention  the first solvent is selected from the group consisting of ethanol  methanol  water  acetonitrile and mixtures thereof. The solvent is a good solvent for carnitine. This means that the solubility of carnitine at room temperature is at least 5%  preferably at least 10% or at least 20% (w/w).Preferably  the first solvent is ethanol. In specific embodiments  the first solvent may comprise up to 0.5%  up to 2% or up to 5% (w/w) water. Therefore  the solvent may be technical grade.

In a preferred embodiment  the solution in step (a) is a saturated solution or an oversaturated solution. It is also preferred that the solution is close to saturation  which means that the carnitine concentration is more than 80% or above 90% of the saturation concentration. In a preferred embodiment of the invention  the concentration of total carnitine in the first solvent is 5 to 75%. In ethanol  the concentration is preferably 10 to 50% or 15 to 40% (w/w). In methanol  the concentration is preferably 20 to 70% (w/w).

Preferably  the carnitine dissolved in the solution in step (a) is pure or highly pure carnitine. In this embodiment  the solution in step (a) only comprises minor amounts of additional substances. Based on the amount of total carnitine (D- and L-) in the solution  the amount of additional substances may be below 0.5  below 1 or below 2% (w/w). In another embodiment  the carnitine dissolved in the solution in step (a) comprises a certain amount of side products or other compounds  such as starting compounds from the production process. In this embodiment  the solution in step (a) may comprise up to 5%  up to 10% or up to 15% (w/w) of other compounds  based on the total amount of carnitine. For example  a synthetic carnitine may comprise less than 1 % (w/w) hydroxycrotonic acid.

In a preferred embodiment  the solution in step (a) is essentially free of water. This means that the carnitine and the solvent should be essentially free of water or comprise as little water as possible. It was found that the method is more efficient when only a low amount of water is present. Preferably  the overall water content in the solution (a) is below 2%  below 1 % or below 0.5% (w/w).

In a preferred embodiment of the invention  step (a) comprises heating the solution  preferably until all the carnitine is dissolved  for example to a temperature above 40°C or above 50°C. Optionally  residual solid carnitine and/or residual solids can be removed subsequently  for example by filtration. In step (a)  the temperature may be adjusted to 40 to 80°C or 50 to 75°C. The temperature is selected depending on the solvent. When ethanol is used  a temperature between 50 and 75°C is preferred  for example around 65°C.

In step (b)  the solution from step (a) can be seeded with L-carnitine crystals. Preferably  the solution which is seeded is a saturated or oversaturated solution. An oversaturated solution is obtainable when preparing in step (a) a saturated solution at elevated temperature and cooling the saturated solution slowly such that no precipitation or crystallization of carnitine occurs.

By seeding the solution with L-carnitine crystals and incubation  growth of the crystals is induced. Thus  when a seeding step (b) is comprised in the inventive process  the average size of the crystals finally obtained is higher. However  carnitine crystals having a high purity at a high yield can also be obtained without adding seeding crystals in a seeding step (b). The crystals essentially consist of L-carnitine or are enriched in L-carnitine. Only low amounts of seeding crystals are necessary for seeding. The seeding crystals should be highly pure and very fine. The seeding crystals should preferably be added when the solution is still clear  i. e. when no or essentially no crystals or precipitates have formed spontaneously yet. This can be achieved by seeding at enhanced temperature. In a preferred embodiment of the invention  in the seeding step (b) the solution has a temperature of 25 to 50°C  preferably between 30 and 45°C. However  during and after seeding and before adding the second solvent  the solubility of the carnitine is still comparably high  and essentially no or only limited rapid formation of crystals is observed.

In a preferred embodiment  the temperature of the solution is reduced after seeding with the crystals. Preferably  the temperature is reduced to 10 to 30°C  for example to about 20°C.

In a step (c)  a second solvent is added  in which the L-carnitine is not soluble or has a low solubility. During and after addition of the second solvent  crystallization of the L-carnitine is observed. During the crystallization of L-carnitine  the solution is depleted of the L-carnitine and becomes a suspension. Thus  after crystallization the composition can be regarded either as a solution or a suspension. When referring to a "solution" with respect to step (b) and consecutive steps  this solution/suspension is meant.

In a preferred embodiment of the invention  the second solvent is selected from acetone  isopropanol  isobutanol  2-propanol  1-pentanol  2-butanone  methylacetate  ethylacetate  butylacetate  tetrahydrofuran  toluene and mixtures thereof. Preferably  the second solvent is acetone.

The second solvent is a solvent  in which L-carnitine is not soluble or only has a low solubility. In a preferred embodiment of the invention  the solubility of L-carnitine in the second solvent is below 3%  below 2% or below 1% (w/w) at 25°C. When adding the second solvent  the overall solubility of carnitine in the solution is decreased  and carnitine is crystallized. Thus upon addition of the second solvent  solid carnitine crystals are formed in the solution.

In a highly preferred embodiment of the invention  the first solvent is ethanol and the second solvent is acetone.

In a preferred embodiment of the invention  in step (c) the ratio of the first solvent to the second solvent is between 1:1 and 1:10 (w/w)  more preferably between 1:1 5 and 1:6 or between 1:2 and 1:4 (w/w). The ratio should be adjusted such that the solubility of the carnitine in the solvent mixture is significantly decreased  such that a high portion of the total carnitine is crystallized.

In a preferred embodiment of the invention  after the optional seeding step (b)  or before or during step (c)  or after adding the second solvent in step (c)  the temperature of the solution is adjusting to between 10°C and 30°C. However  the second solvent can be added before or after reducing the temperature of the solution.

Preferably  the second solvent in step (c) is added slowly  for instance drop wise. In a preferred embodiment of the invention  in step (c) the second solvent is added to the solution for 20 min to 8 h  or for 1 h to 6 h. During the addition of the acetone and incubation  L-carnitine is crystallized. The amount of L-carnitine crystallized usually can be increased by incubation at low temperature.

Preferably  after the addition of the second solvent the composition is incubated at a reduced temperature. For example  the temperature may be reduced to 5 to 20°C  or below 15°C. The composition may be incubated at this temperature for 10 min to 2 days  or for 30 minutes to 24 hours. In a preferred embodiment of the invention  after the addition of the second solvent in step (c) the composition is incubated for 10 min to 2 days at a temperature below 20°C.

In step (d)  the solid crystals are isolated by known means  for instance by filtration or sedimentation. Optionally  the crystals are washed  preferably with the second solvent. The solvent is removed by drying  optionally at reduced pressure. Fort instance  acetone may be removed by drying at 55°C at a pressure below 100 mbar.
According to the invention  the total solid carnitine isolated in step (d) is referred to as "crystals". The solid was found to have a crystalline structure. However  especially upon rapid addition of the second solvent  the solid carnitine "crystals" might also comprise  at least in part  a carnitine precipitate.
The overall yield of L-carnitine  based on the total L-carnitine in the starting solution  is preferably above 80% or above 85%.

In a preferred embodiment of the invention  the method comprises the steps of
(a) providing a solution comprising at least 5% (w/w) carnitine in ethanol  wherein the carnitine comprises at least 50 % (e.e.) L-carnitine  wherein the solution is heated until all the carnitine is dissolved 
(a1) adjusting the temperature of the solution to 25 to 50°C 
(b) seeding the solution with L-carnitine crystals
(b1) optionally adjusting the temperature to 10 to 30°C 
(c) adding acetone in an amount such that the ratio ethanol/acetone is between 1:1 and 1:10 (w/w) 
(c1) optionally cooling the composition to a temperature below 20°C 
(d) isolating crystals comprising L-carnitine  preferably by filtration.

In the method recited above  steps (a) to (d) are carried out consecutively.

In a specific embodiment of the invention  the overall method of the invention is repeated with the crystals obtained in step (d). In this embodiment  a solution of crystals in a first solvent is prepared. When repeating the overall process twice or more  a highly pure L-carnitine is obtainable even from a carnitine of low enantiomeric purity.

The inventive process solves the above-mentioned problem. A highly pure L-carnitine is obtainable from a mixture of D- and L-carnitine by the inventive process. When separating the mixture of D- and L-carnitine  it is not necessary to add optically active compounds  as for example disclosed in DE 35 36 093 or DD 93 347. In the inventive process  the carnitine is not converted to an optically active salt in an intermediate step. It was surprising that the inventive process is highly efficient  because it was generally assumed in the art that an effective separation of a zwitterion is difficult without optically active additives (see DD 93 347  column 2  lines 8-11). It thus could not be assumed that by the relatively simple process of the invention a high increase of the L-enantiomeric form would be obtained.

The inventive method is applicable in a simple manner with a low number of process steps. The process does not require the precipitation of optically active salts of L-carnitine  isolation and decomposition by additional crystallization steps. The process provides L-carnitine at high enantiomeric purity and also at high yield. The process is less cost- and labour-intensive  compared to processes known in the art. It can be used to purify L-carnitine from enantiomeric mixtures  for instance those obtained by industrial synthesis processes.

Examples

Example 1

A laboratory reactor is charged with 100g of carnitine and 300g of ethanol. The reactor is heated up to 65°C and stirred until all carnitine has been dissolved. Afterwards the reactor temperature is set to 37°C. At 37°C seed crystals of pure L-carnitine are added. The reactor temperature is cooled down to 20°C at a rate of -0.2K/min. At 20°C 900g of acetone are added within 2 hours. Afterwards the suspension is cooled down to 10°C. At 10°C the solids are isolated and washed with acetone and dried at 55°C and <100mbar.

As a result  86.1 g of a crystalline-white dry solid were obtained. The solid comprised 99.036% (w/w) of total carnitine. The enantiomeric purity was 99.60% (e.e.). The residual solvent content was 349 mg/kg ethanol and 386 mg/kg acetone. The total yield of L-carnitine was 88.6%.

Example 2

A laboratory reactor is charged with 60.2g of carnitine and 60g of methanol. The reactor is heated up to 50°C and stirred until all carnitine has been dissolved. Afterwards the reactor temperature is set to 25°C. At 25°C 0.74g of seed crystals of pure L-carnitine are added. The reactor temperature is cooled down to 20°C at a rate of -0.2K/min. At 20°C 180g of acetone are added within 1 hour. Afterwards the suspension is cooled down to 10°C within 50 min. The suspension is stirred at this temperature for another 30min. Afterwards the solids are filtered via a Nutsch filter and washed twice with approximately 60g of acetone and subsequently dried for 8h at 55°C and a pressure of 250 mbar.

As a result  45.78 g of a crystalline-white dry solid were obtained. The solid comprised 98.94% (w/w) of total carnitine. The enantiomeric purity was 99.78% (e.e.).

Example 3

A laboratory reactor is charged with 30.1g of carnitine and 90g of ethanol. The reactor is heated up to 65°C and stirred until all carnitine has been dissolved. Afterwards the reactor temperature is set to 37°C. At 37°C 0.91g of seed crystals of pure L-carnitine are added. The reactor temperature is cooled down to 20°C at a rate of -0.2K/min. At 20°C 270g of ethyl acetate are added within 1 hour. Afterwards the suspension is cooled down to 10°C within 50 min. The suspension is stirred at this temperature for another 30min. Afterwards the solids are filtered via a Nutsch filter and washed twice with approximately 30g of ethyl acetate and subsequently dried for 8h at 55°C and a pressure of 250 mbar.

As a result  28.66 g of a crystalline-white dry solid were obtained. The solid comprised 98.51% (w/w) of total carnitine. The enantiomeric purity was 99.46% (e.e.).

WE CLAIM:

1. A method for the production of L-carnitine  comprising the steps of
(a) providing a solution comprising at least 5% (w/w) carnitine in a first solvent  wherein the carnitine is a mixture of D- and L-carnitine 
(b) optionally seeding the solution with L-carnitine crystals 
(c) adding a second solvent  in which the L-carnitine is not soluble or has a low solubility 
(d) isolating crystals comprising L-carnitine.
2. The method of at least one of the preceding claims  wherein the first solvent is selected from the group consisting of ethanol  methanol  water  acetonitrile and mixtures thereof.
3. The method of at least one of the preceding claims  wherein in step (a) the concentration of total carnitine in the first solvent is 5 to 75% (w/w).
4. The method of at least one of the preceding claims  wherein in step (a) the carnitine comprises more than 80% (e.e.) L-carnitine and in step (d) the crystals comprise more than 95% (e.e.) L-carnitine.
5. The method of at least one of the preceding claims  wherein step (a) comprises heating the solution to a temperature of above 40°C.
6. The method of at least one of the preceding claims  wherein the second solvent is selected from acetone  isopropanol  isobutanol  2-propanol  1-pentanol  2-butanone  methylacetate  ethylacetate  butylacetate  tetrahydrofuran  toluene and mixtures thereof.
7. The method of at least one of the preceding claims  wherein the solubility of L-carnitine in the second solvent is below 2% (w/w) at 25°C.
8. The method of at least one of the preceding claims  wherein in the seeding step (b) the solution has a temperature of 25 to 50 °C.
9. The method of at least one of the preceding claims  wherein in step (c) the ratio of the first solvent to the second solvent is between 1:1 and 1:10 (w/w).
10. The method of at least one of the preceding claims  wherein after the seeding step (b)  before or during step (c)  or after adding the second solvent in step (c)  the temperature of the solution is adjusted to between 10°C and 30°C.
11. The method of at least one of the preceding claims  wherein in step (c) the second solvent is added to the solution during a time span of 20 min to 24 h.
12. The method of at least one of the preceding claims  wherein after the addition of the second solvent in step (c) the composition is incubated for 10 min to 2 days at a temperature below 20°C.
13. The method of at least one of the preceding claims  wherein in step (d) the crystals comprise more than 99% (e.e.) L-carnitine.
14. The method of at least one of the preceding claims  wherein in step (d) the crystals are isolated by filtration or sedimentation.
15. The method of at least one of the preceding claims  wherein the first solvent is ethanol and the second solvent is acetone.