METHOD FOR REMOVING ANTIFOAMING AGENTS FROM A FERMENTATION BROTH

Abstract

The present invention relates to a method for separating a molecule of interest from an antifoaming agent in a fermentation broth comprising said molecule of interest and said antifoaming agent. The method comprises the steps of filtering the fermentation broth at a temperature in the range of 1° C. to 15° C. above the cloud point of the antifoaming agent, and thereby obtaining a first fraction of the fermentation broth comprising the molecule of interest and a second fraction of the fermentation broth comprising the antifoaming agent, wherein the antifoaming agent is a polyalkylene glycol based antifoaming agent. The invention further relates to a process for purifying a molecule of interest from a fermentation broth comprising said method.

Claims

1. A method for separating a molecule of interest from an antifoaming agent in a fermentation broth comprising the molecule of interest and the antifoaming agent, the method comprising: (a) filtering the fermentation broth at a temperature in the range of 1° C. to 15° C. above the cloud point of the antifoaming agent; and (b) thereby obtaining a first fraction of the fermentation broth comprising the molecule of interest and a second fraction of the fermentation broth comprising the antifoaming agent, wherein the antifoaming agent is a polyalkylene glycol based antifoaming agent.

2. The method according to claim 1, wherein the antifoaming agent has a cloud point between 15° C. and 40°.

3. The method according to claim 1, wherein the temperature in step (a) in the range of 1° C. to 15° C. above the cloud point of the antifoaming agent, is a temperature in the range of 20° C. to 45° C.

4. The method according to claim 1, wherein the antifoaming agent is selected from the group consisting of a polypropylene glycol (PPG), a polyethlyene glycol (PEG), polyalkylene glycol (PAG), an ethylene/propylene oxide block copolymer, a polyalcohol based on EO/PO block copolymer, an alkoxylated fatty acid ester, a polypropylene-polyethylene glycol copolymer (PPG-PEG), mixtures thereof, and derivatives thereof.

5. The method according to claim 1, wherein the antifoaming agent is selected from the group consisting of PPG, PEG, and PPG-PEG.

6. The method according to claim 1, wherein the filtering in step (a) comprises microfiltration.

7. The method according to claim 1, further comprising a step (c) of subjecting the first fraction of the fermentation broth comprising the molecule of interest obtained in step (b) to ultrafiltration, diafiltration, and/or dia-ultrafiltration at a temperature below the cloud point of the antifoaming agent.

8. The method according to claim 1, wherein the molecule of interest is an enzyme.

9. The method according to claim 1, wherein the molecule of interest is an enzyme selected from the group consisting of amylase, alpha-amylase, glucoamylase, pullulanase, protease, lipase, cutinase, acyl transferase, cellulase, endoglucanase, glucosidase, cellubiohydrolase, lactase, xylanase, xyloglucantransferase, xylosidase, mannanase, phytase, phosphatase, xylose isomerase, glucoase isomerase, acetolactate decarboxylase, pectinase, pectin methylesterase, polygalacturonidase, lyase, pectate lyase, arabinase, arabinofuranosidase, galactanase, laccase, peroxidase, and an asparaginase.

10. The method according to claim 1, wherein the fermentation broth comprises recombinant or non-recombinant microbial cells capable of producing the molecule of interest.

11. The method according to claim 1, wherein prior to step (a) the fermentation broth is obtained by cultivating microbial cells in a fermentation medium comprising the antifoaming agent.

12. The method according to claim 1, wherein step (a) comprises cross flow filtration or dead-end filtration.

13. The method according to claim 10, wherein the molecule of interest is secreted by the microbial cells into the fermentation broth.

14. A process for purifying a molecule of interest from a fermentation broth comprising the method steps according to claim 1.

15. The process according to claim 14, further comprising a step of further purifying the molecule of interest, by ion exchanging the first fraction of the fermentation broth of step (b) comprising the molecule of interest.

16. The process according to claim 14, further comprising a step of preparing a formulation containing the molecule of interest.

17. The method according to claim 5 wherein the antifoaming agent is a PPG having an average molecular mass in the range of 500 to 5000 g/mol.

18. The method according to claim 7 wherein the temperature below the cloud point of the antifoaming agent is 2° C. to 18° C.

19. The method according to claim 10 wherein the microbial cells are bacteria cells.

Description

EXAMPLES

[0130] The invention will now be illustrated by working Examples. Theses working Examples must not construed, whatsoever, as limitations of the scope of the invention.

[0131] Unless otherwise stated the following experiments have been performed by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering and fermentative production of chemical compounds by cultivation of microorganisms. See also Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y, 1989) and Chmiel et al. (Bioprocesstechnik 1. Einführung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991).

[0132] The fermentation broths for the examples below (Examples 1-3) were obtained by culturing Bacillus licheniformis cells comprising a gene coding for a subtilisin protease. Bacillus licheniformis cells were cultivated in a fermentation process using a chemically defined fermentation medium providing the components listed in Table 1 and Table 2.

TABLE-US-00001 TABLE 1 Macroelements provided during the course of the fermentation process Concentration [g/L initial Compound Formula volume] Citric acid C.sub.6H.sub.8O.sub.7 3.0 Calcium sulfate CaSO.sub.4 0.7 Monopotassium phosphate KH.sub.2PO.sub.4 25 Magnesium sulfate MgSO.sub.4*7H.sub.2O 4.8 Sodium hydroxide NaOH 4.0 Ammonia NH.sub.3 1.3

TABLE-US-00002 TABLE 2 Trace elements provided during the course of the fermentation process Trace Concentration element Symbol [mM] Manganese Mn 24 Zinc Zn 17 Copper Cu 32 Cobalt Co 1 Nickel Ni 2 Molybdenum Mo 0.2 Iron Fe 38

[0133] A solution containing 50% glucose was used as feed solution. pH was adjusted during fermentation using ammonia. At the desired product titer the fermentation was terminated, and the product amylase was present in both soluble and crystalline form as confirmed by visual inspection using a microscope. At the end of fermentation, the phosphate concentration was about 3 g/L.

Example 1: Experimental Determination of Cloud Point of Pluriol® P2000 (a PPG Having an Average Molar Mass of Approx. 2000 g/Mol)

[0134] 600 mL of deionized water was cooled to about 5° C. in an ice bath and a corresponding amount of Pluriol® P2000 was added to adjust its concentration to 0.1, 1.0 or 2.5 g/L. The solution was emulsified using a Polytron homogenizer at 7500 rpm for 10 min. After a few minutes the resulting air bubbles disappeared and the clear solution was slowly warmed under constant gentle stirring. Samples (15 mL) were taken at certain temperature points and the turbidity was measured using a portable turbidimeter 2100Q from Hach turbidity. A strong increase in turbidity as a function of temperature indicated the observed cloud point temperature (Tcp).

TABLE-US-00003 TABLE 3 Concentration of Pluriol ® Observed P2000 Top [g/L] [° C.] 0.1 27-28 1.0 22-23 2.5 20-21

Example 2: Microfiltration of a Fermentation Broth of B. licheniformis Comprising Antifoaming Agent at Various Temperatures

[0135] The antifoaming agent present in the fermentation broth was Pluriol® P2000 (a polypropylene glycol commercially available from BASF) with a cloud point in the range of 20° C. to 27° C. as determined in example 1 above. Dead-end microfiltration was performed using a 0.2 μm filter and device from Pall Corporation Pall Filter Supor EKV membrane—KA3EKVP6G. Various fractions of a fermentation broth of B. licheniformis, biomass-free, were subjected to microfiltration. Although post-processing steps for removing the biomass were already conducted, the amount of antifoaming agents was still significant (see Table 4).

[0136] The amount of antifoaming agent in the respective fractions was determined at room temperature. The amount of the antifoaming agent was determined by H PLC-MS analysis using a Single Quad MS LCMS device. The device was a Shimadzu Nexera; Single-Quad MS LCMS2020; the HPLC column was a Kinetex C18; 2.1×100 mm; 1.7 μm; the solvent was A: water; 0.1% HCOOH and B: acetonitrile/isopropanol (50/50); 0.1% HCOOH; the quantification was performed using the triple charged species of the ten highest signals.

TABLE-US-00004 TABLE 4 Anti- foaming agent Fraction [mg/ml] Batch Starting material: fermentation broth, 1.46 1-1 biomass-free Filtrate obtained by filtering at 6 to 15° C. 1.12 Batch Starting material: fermentation broth, 1.64 1-2 biomass-free Filtrate obtained by filtering at 35° C. 0.44 Batch Starting material: fermentation broth, 0.44 2-1 biomass-free Filtrate obtained by filtering at 6 to 15° C. 0.46 Batch Starting material: fermentation broth, 0.42 2-2 biomass-free Filtrate obtained by filtering at 35° C. 0.18

[0137] It can be seen that filtration at a temperature above the cloud point of Pluriol® P2000, e.g. at 35° C., leads to efficient depletion of residual antifoaming agent from the fermentation broth, while filtration at lower temperatures, e.g. 6° C. to 15° C., has only marginal effects on the amount of residual antifoaming agent in the fermentation broth.

Example 3: Depletion of Antifoaming Agent and Biomass from a Fermentation Broth of B. licheniformis Comprising Protease

[0138] Pluriol® P2000 was used as the antifoaming agent. The amount of antifoaming agent was determined as in example 2. The fermentation broth was centrifuged in a laboratory centrifuge at 10000 g for 10 min to obtain the supernatant.

[0139] This example demonstrates that the antifoaming agent can be successfully removed or at least largely depleted from the fermentation broth during biomass separation. Hence, biomass and antifoaming agent may be removed together.

TABLE-US-00005 TABLE 5 Total anti- Anti- Batch vol. Explanation foaming Temp. of foaming after prec. concerning agent in prec. DSP st. agent DSP st. vol. of sample Sample [° C.] [mg/ml] [kg] sample [g] Supernatant of 30 3.1 12 prior to MF 37.2 fermentation (fermentation) broth Pool permeate 30 (MF) 0.057 46.74 vol. of 2.66 after MF permeate after MF Pool retentate 10 (UF) 0.142 0.86 after 0.12 after UF (prior to concentrating diafiltration, after concentrating) Pool retentate 10 (UF) 0.066 0.57 washed with 0.04 after dia-UF 1.49 kg, concentrated prec. DSP st.-preceding downstream processing step vol.-volume

[0140] Performing the microfiltration (MF) at a temperature above the CP (e.g. at 30° C.) results in efficient removal of the major part of the antifoaming agent. The permeate of the microfiltration comprises the molecule of interest (enzyme) and significantly less amount of antifoaming agent compared to the fermentation broth. Subsequent ultrafiltration (UF) and dia-ultrafiltration at a temperature below the CP of the antifoaming agent results in further depletion of the residual antifoaming agent together with the permeate, while the enzyme retains in the retentate and may be subjected to further purification steps such as chromatography or a second ultrafiltration. The amounts of antifoaming agents in the permeate of the microfiltration and in the ultrafiltration-retentate are significantly reduced. As a further advantage, fouling of the ultrafiltration membrane and during chromatography in subsequent purification steps can be avoided.