WO2008125616A2

WO2008125616A2 - Method for the purification of bio-molecules

Info

Publication number: WO2008125616A2
Application number: PCT/EP2008/054407
Authority: WO
Inventors: Van De Emilius Johannes Albertus Xaverius Sandt; Aad Johannes Bouman; De Erik Vos Burchart
Original assignee: Dsm Ip Assets B.V.
Priority date: 2007-04-12
Filing date: 2008-04-11
Publication date: 2008-10-23
Also published as: WO2008125616A3

Abstract

The present invention provides a method for purifying a compound of interest comprising the steps of: i.) Adjusting the pH of an aqueous mixture comprising biomass and said compound of interest to a value at which the amount of dissolved compound of interest is less than 10% of the total amount of said compound of interest present in said mixture; ii.) Isolating a slurry from the mixture obtained in step i.) by removing at least 40% of the water present in the mixture obtained in step L); iii.) Adjusting the pH of the slurry obtained in step ii.) to a value at which the amount of dissolved compound of interest is more than 90% of the total amount of said compound of interest present in said slurry; iv.) Removing solids from the mixture obtained in step iii.) to give a solution comprising said compound of interest, wherein said removing in steps ii.) and iv.) is carried out with the same means.

Description

METHOD FOR THE PURIFICATION OF BIO-MOLECULES

Field of the invention

The present invention relates to purification of compounds obtained in fermentation processes.

Background of the invention

Purification and isolation are technical measures that are comprised in almost any production process of compounds of interest (COI's), since production processes only very rarely produce the desired COI in the required purity and/or physical state without these measures. The requirement for purification and isolation is particularly pronounced for bio-molecules that are produced in fermentation processes based on complex microorganisms or plants and the like. In these cases substantial amounts of unwanted components are released during fermentation and downstream processing. These components can vary widely in nature, ranging from relatively simple chemical structures such as amino acids to complex structures such as cell wall debris. The production of COI's in microorganisms is becoming increasingly important as it usually presents an environmentally safe alternative to chemical synthesis. Not only does this type of production require breakthrough technologies within the cell, it also poses great challenges outside the cell, in down stream processing and isolation.

There are many examples of purification and isolation processes. In most cases, the microorganism used for production needs to be separated from the COI. When the COI is produced intra-cellular the cell walls must be disrupted prior to separation. When the COI is produced under conditions wherein the COI is undissolved, the COI is usually brought in solution. For the actual separation a wide variety of techniques is available to the person skilled in the art, such as centrifugation, filtration and sedimentation. The COI in question normally has to be separated from impurities, again by standard techniques such as extraction, reversed osmosis, ultra filtration, chromatography and the like and most frequently by combinations thereof. Finally the COI is isolated by crystallization, precipitation or the like.

Examples of COI's produced along the above described methodology are manifold. These COI's can be classified in different ways, for instance by acidity (acids such as fusidic acid, mycophenolic acid or penicillin G and bases such as erythromycin) or by hydrophilicity (hydrophobic compounds such as ceramides, lipstatin, polyunsaturated fatty acids, rapamycin or tacrolimus and hydrophilic compounds such as cephalosporin C or daunorubicin) and of course each class represents its own specific purification and isolation characteristics that can often be successfully predicted by the person skilled in the art.

The prior art procedures mentioned above require a multitude of steps and, on a production scale level, various expensive equipment. Purification and isolation procedures comprising less steps and equipment are therefore extremely attractive.

Detailed description of the invention

The term "permeate" refers to the part of a mixture that, upon separation using a certain separation technique, passes through the separation means. In case of separation techniques such as (membrane) filtration, micro filtration or ultra filtration "permeate" refers to the part of the mixture that passes through the openings of the filter or membrane. In case of centrifugation or sedimentation, "permeate" refers to the part of the mixture that has the lowest density.

The term "retentate" refers to the part of a mixture that, upon separation using a certain separation technique, does not pass through the separation means. In case of separation techniques such as cloth filtration, membrane filtration, micro filtration or ultra filtration "retentate" refers to the part of the mixture that does not pass through the openings of the filter or membrane. In case of centrifugation or sedimentation, "retentate" refers to the part of the mixture that has the highest density. The term "slurry" refers to a mixture of solids and liquids. Examples of solids within the scope of the present invention are particles, such as for instance crystals or amorphous particles of a COI, cell walls and parts of cell walls. Examples of liquids within the scope of the present invention are water, aqueous solutions comprising dissolved COI's, enzymes, amino acids, sugars, salts and the like. In a first aspect of the present invention there is provided a method for purifying a COI, following the production of said COI by a microorganism or a plant, comprising of the following steps. In a first step the pH of the resulting broth comprising biomass and COI is adjusted to a value at which the solubility of the COI is relatively low; with this is meant a solubility whereby the amount of dissolved COI is less than 20%, preferably less than 15%, more preferably less than 10%, most preferably between 0.01% and 5% of the total amount of said COI present in said mixture. A lower solubility will in general result in a higher separation yield. The pH value mentioned above is dependent on the chemical nature of the COI (Ae. compounds with low pKa values require low pH values, whereas compounds with high pKa values require high pH values). Optionally, if the pH value of the broth already has the required value, adjustment is not necessary.

In a second step, the resulting slurry comprising biomass and, mostly undissolved, COI is then subjected to separation. Suitable separation techniques are centrifugation, (cloth) filtration, micro filtration, sedimentation, ultra filtration and the like. Preferably, the separation is carried out in such a way that at least 40%, more preferably 60%, most preferably 75%, still most preferably 90%, of the water present in the slurry is transferred to the permeate. Optionally, the retentate is washed with a washing liquid which results in a further lowering of the amount of unwanted components. Preferably this washing liquid is water or an aqueous solution or an aqueous buffering system, more preferably the pH of this washing liquid is adjusted to a value deviating not more than 0.5 units from the pH of the retentate.

In a third step, the pH value of the retentate is then adjusted to a value at which the amount of dissolved COI is more than 90%, preferably more than 95%, more preferably more than 98%, most preferably from 99% to 99.99% of the total amount of said COI present in the retentate.

In a fourth step, the resulting slurry comprising biomass and, mostly dissolved, COI is then subjected to separation. Suitable separation techniques are centrifugation, filtration, micro filtration, sedimentation, ultra filtration and the like. Preferably, the separation is carried out in such a way that at least 50%, more preferably 70%, most preferably 90%, still most preferably 95%, of the liquid present in the slurry is transferred to the permeate. Optionally, the retentate is washed with a washing liquid which results in increased yield of COI. Preferably this washing liquid is water, more preferably the pH of this washing liquid is adjusted to a value deviating not more than 0.5 units from the pH of the permeate. Advantageously, the separation technique in the fourth step is the same as that used in the second step and preferably the separation medium (centrifuge, filter, filter cloth, membrane and the like) is also the same as that used in the second step. This reduces the amount of equipment, unit operations and points where errors can occur and thus facilitates process control compared to prior art methods, such as for instance WO 2005/105768, where a first separation technique (Ae. filtration) is followed by a second technique (Ae. extraction).

Adjustment of the pH in the above mentioned steps of the first aspect of the invention can be carried out using any mineral or organic acids (some suitable examples are HCI, H₂SO₄, HNO₃, H₃PO₄, acetic acid, citric acid, succinic acid and the like) or bases (some suitable examples are KOH, LiOH, NaHCO₃, NaOH, NH₄OH, pyridine, triethylamine, morpholine and the like).

In a first embodiment, the cell walls of the microorganism are treated using techniques known to the person skilled in the art with the objective of releasing intracellular^ produced COI, prior to the first step of the method of the present invention. Such a treatment (Ae. lysis) greatly enhances the overall yield of the method.

In a second embodiment, the COI is a compound having a pKa value lower than 6, preferable ranging from 3 to 5.5, more preferably from 3.5 to 5, most preferably from 4 to 4.7. In the first step mentioned above, the pH of the broth comprising biomass and COI is adjusted to a value ranging from 0.1 to 7, preferably from 1 to 6, more preferably from 2 to 5.5, most preferably from 3 to 5, still most preferably from 3.5 to 4.7. In the third step mentioned above, the pH of the retentate comprising biomass and COI is adjusted to a value ranging from 7 to 14, preferably from 7.5 to 12, more preferably from 8 to 11 , most preferably from 8.5 to 10.

A COI suitable as example for purification according to the second embodiment is mycophenolic acid (MPA), an immunosuppressant having a pKa value of 4.5. MPA can be obtained by fermentation of Penicillium brevicompactum, Penicillium stoloniferum and related species, for instance as described in US 4,452,891. During MPA fermentation many impurities are formed, amongst which structures, as described in the European Pharmacopoeia, which are closely related to MPA itself. Using the method described in the first aspect, a retentate is obtained from which MPA can be isolated in high purity. One of the problems associated with fermentation of MPA is the formation of an unwanted side product, (4E)-6-(1 ,3-dihydro-1 ,4-dihydroxy-6-methoxy-7-methyl-3-oxo- 5-isobenzofuranyl)-4-methyl-4-hexenoic acid (hydroxylated MPA). The method of the present invention is particularly suitable for lowering the amount of this impurity in the final MPA product without having to use cumbersome purification techniques such as chromatography or (re-)crystallization. Other COI's suitable for purification according to the second embodiment are carboxylic acids such as abietic acid, acetoxolone, alazopeptin, amphotericin, arachidic acid, arachidonic acid, atorvastatin, bilirubin, biotin, candicidin, chaulmoogric acid, cholic acid, chrysanthemic acid, clavulanic acid, docosahexaenoic acid, eicosapentaenoic acid, fluvastatin, frenolicin, fumagillin, fusidic acid, helvolic acid, indoleacetic acid, jasmonic acid, lactobacillic acid, laidlomycin, leptomycin B, levopimaric acid, linoleic acid, linolenic acid, lisonopril, lucensomycin, lysergic acid, montelukast, mupirocin, N-acetyl muramic acid, nodulisporic acid, pantothenic acid, penicillin G, penicillin V, perindopril, pimaric acid, prostacyclin, prostaglandin, pseudomonic acid, quinapril, ramipril, repaglinide, retinoic acid, rifamycin B, thermorubin, thromboxane, trandolapril, vernolic acid and the like. In a third embodiment, a solvent is added to facilitate dissolution of the COI during the third step of the method of the present invention. Such a solvent can be any water-miscible solvent. Preferably such a solvent is water or an aqueous solution or an aqueous buffering system, more preferably the pH of this washing liquid is adjusted to a value deviating not more than 0.5 units from the pH of the retentate, meaning the pH value obtained after adjustment.

In a fourth embodiment, the COI is isolated from the permeate obtained in the fourth step. Preferably this is done by means of crystallization, extraction, freeze-drying or precipitation. Crystallization and precipitation can be effected by changing the pH value and/or the temperature and/or adding solvents to the precipitate.

In a second aspect of the present invention the COI obtained by the method of the first aspect can be used for the preparation of another COI. This can be done by any means available to the person skilled in the art, such as enzymatic or chemical conversion or a combination thereof. In a first embodiment, an acid such as MPA can be esterified with a primary alcohol such as 2-morpholinoethanol, to give a product, in this case mycophenolate mofetil (MPM). MPM has therapeutic relevance as a prodrug of MPA. Esterification can be carried out using any of the many methods available for the person skilled in the art. EXAMPLES

General Methods

HPLC analysis was performed on a Waters Atlantis dC₁₈ column (5μm; 4.6x150 mm; W 32371 X 12), using as mobile phase A MiIIiQ water with 0.1 vol% formic acid and as mobile phase B CH₃CN with 0.1 vol% HCO₂H. The run time was 13 minutes and the flow 1.5 mL.min^"1. Detection wavelength was at 251 nm. The following gradient was applied:

As dilution buffer 400 mg ammonium formate was dissolved in 700 ml. water and the pH was adjusted to 3.5 with HCO₂H and next 1300 ml. acetonitrile was added. In the above system, the retention time of mycophenolic acid was 4.9 min.

Example 1 Purification of mycophenolic acid using pH-shifted membrane filtration

The pH of a fermentation broth resulting from a fed-batch Penicillium brevicompactum- based fermentation process (2.0 L, containing 2.8 g/L mycophenolic acid, MPA, prepared as described in US 4,452,891 ) was adjusted to 6.2 with H₂SO₄ (6N). At 20±3°C, the broth was then concentrated with ultra filtration on a 50 NM ceramic Membralox membrane to a volume of 1.2 L. After this concentration step the concentrated broth was washed with water (1.25 L). Several impurities have been washed out at this pH, however not (4E)-6-(1 ,3-dihydro-1 ,4-dihydroxy-6-methoxy-7- methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoic acid (hydroxylated MPA, remains at 9%). HPLC analysis indicated that the permeate contains less than 50% of the MPA. The pH of the concentrated broth was then adjusted to 8.5 with NaOH (4N) and the broth was washed out through the membrane with water (1.18 L) to transfer pure MPA to the permeate in a yield of 25%. Example 2 Purification of mycophenolic acid using pH-shifted membrane filtration at low pH

The pH of a fermentation broth resulting from a fed-batch Penicillium brevicompactum- based fermentation process (1.5 L, containing 2 g/L MPA, prepared as described in US 4,452,891 ) was adjusted to 4.5 with H₂SO₄ (6N). At 20±3°C, the broth was then concentrated with ultra filtration on a 50 NM ceramic Membralox membrane to a volume of 500 ml_. After this concentration step the concentrated broth was washed with water (2 L). Several impurities have been washed out at this pH, especially hydroxylated MPA (from 1 1% down to 6%). HPLC analysis indicated that the permeate contains less than 10% of the MPA. The pH of the concentrated broth was then adjusted to 8.5 with NaOH (4N) and the broth was washed out through the membrane with water (1.5 L) to transfer pure MPA to the permeate in a yield of 83%.

Example 3 Purification of mycophenolic acid using pH-shifted membrane filtration at low pH and elevated temperature

The pH of a fermentation broth resulting from a fed-batch Penicillium brevicompactum- based fermentation process (3.0 L, containing 2 g/L MPA, prepared as described in US 4,452,891 ) was adjusted to 4.0 with H₂SO₄ (6N). At 50±2°C, the broth was then concentrated with ultra filtration on a 50 NM ceramic Membralox membrane to a volume of 1.0 L. After this concentration step the concentrated broth was washed with water (2 L). Several impurities have been washed out at this pH, especially hydroxylated MPA (from 1 1% down to 5%). HPLC analysis indicated that the permeate contains less than 10% of the MPA. The pH of the concentrated broth was then adjusted to 8.5 with NaOH (4N) and the broth was washed out through the membrane with water (3.0 L) to transfer pure MPA to the permeate in a yield of 90%.

Claims

1. Method for purifying a compound of interest comprising the steps of: i.) Adjusting the pH of an aqueous mixture comprising biomass and said compound of interest to a value at which the amount of dissolved compound of interest is less than 10% of the total amount of said compound of interest present in said mixture; ii.) Isolating a slurry from the mixture obtained in step i.) by removing at least

40% of the water present in the mixture obtained in step L); iii.) Adjusting the pH of the slurry obtained in step ii.) to a value at which the amount of dissolved compound of interest is more than 90% of the total amount of said compound of interest present in said slurry; iv.) Removing solids from the mixture obtained in step iii.) to give a solution comprising said compound of interest, characterized in that said removing in steps ii.) and iv.) is carried out with the same means.

2. Method according to claim 1 further comprising washing the slurry of solids obtained in step ii.) with an aqueous solution having a pH value deviating not more than 0.5 units from the adjusted pH value of step i.) and/or washing the solids after step (iv.) with an aqueous solution having a pH value deviating not more than 0.5 units from the adjusted pH value of step iii.).

3. Method according to anyone of claims 1 to 2 further comprising isolating said compound of interest from said solution obtained in step iv.) by means of concentration and/or crystallization and/or precipitation and/or extraction.

4. Method according to anyone of claims 1 to 3 wherein said compound of interest is a compound having a pKa value lower than 6.

5. Method according to claim 4 wherein said compound is chosen from the list consisting of abietic acid, acetoxolone, alazopeptin, amphotericin, arachidic acid, arachidonic acid, atorvastatin, bilirubin, biotin, candicidin, chaulmoogric acid, cholic acid, chrysanthemic acid, clavulanic acid, docosahexaenoic acid, eicosapentaenoic acid, fluvastatin, frenolicin, fumagillin, fusidic acid, helvolic acid, indoleacetic acid, jasmonic acid, lactobacillic acid, laidlomycin, leptomycin B, levopimaric acid, linoleic acid, linolenic acid, lisonopril, lucensomycin, lysergic acid, montelukast, mupirocin, N-acetyl muramic acid, mycophenolic acid, nodulisporic acid, pantothenic acid, penicillin G, penicillin V, perindopril, pimaric acid, prostacyclin, prostaglandin, pseudomonic acid, quinapril, ramipril, repaglinide, retinoic acid, rifamycin B, thermorubin, thromboxane, trandolapril and vernolic acid.

6. Method according to claim 5 wherein said compound is mycophenolic acid.

7. Method according to anyone of claims 4 to 6 wherein said value of said pH in step i.) is from 0.1 to 6.5.

8. Method according to anyone of claims 4 to 7 wherein said value of said pH in step iii.) is from 7.5 to 14.0.

9. Method according to anyone of claims 1 to 8 wherein said removing in step ii.) and/or step iv.) is carried out by centrifugation, filtration, micro filtration or ultra filtration.

10. Use of a compound of interest obtained according to anyone of claims 1 to 9 for the preparation of another compound of interest.