Method for producing agar or agarose beads using natural or vegetable oils

Abstract

A method for producing agar or agarose beads, comprising the steps of: (i) preparing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature higher than the gelling temperature of the aqueous solution; (ii) preparing an oil phase comprising a natural or vegetable oil at a temperature higher than the gelling temperature of the aqueous solution prepared in step (i); (iii) mixing the aqueous phase prepared in step (i) with the oil phase obtained in step (ii) in a reactor and adding an emulsifier; and (iv) emulsifying the mixture obtained in step (iii), preferably by stirring the mixture, thereby forming an emulsion. v) performing stepwise cooling of the emulsion obtained in step (iv) comprising a first cooling step for cooling the emulsion to a temperature 0.1-30° C. higher than the gelling temperature of the aqueous solution obtained in step (i) followed by a second cooling step for cooling the emulsion to a temperature lower than the gelling temperature of the aqueous solution prepared in step (i) by emptying the emulsion from the reactor and passing the emulsion through a heat exchanger; and (vi) recovering the agar or agarose beads from the emulsion.

Description

[Technical field]

[0001] The present invention relates generally to a method for producing agar or agarose beads suitable for use as a chromatography resin by emulsifying a mixture using natural or vegetable oils. [Background technology]

[0002] Agarose beads for separation purposes have been commercially available for 50 years. Traditionally, agarose beads are obtained by emulsifying a warm solution of agarose in a warm water-immiscible solvent to form a water-in-oil (W / O) emulsion, followed by cooling the emulsion below the gelling temperature of agarose to form bead particles. These are collected in a subsequent separation step. Such a process has been previously described, for example, in PORATH JET AL, Journal of Chromatography, vol. 60, January 1, 1971, US2018071484 and WO1989011493.

[0003] An important parameter of agarose beads used as chromatography resins is their porosity. Porosity is controlled by various parameters during manufacture, with cooling of the emulsified solution being one of the most important parameters. Controlling the cooling of the emulsified mixture and the temperature gradient during cooling is essential to achieve the desired range of porosity of the beads.

[0004] Traditionally, the water-immiscible solvent used as the continuous phase of the emulsion is usually selected from organic solvents, such as toluene. Toluene has an advantage in that it has a good control of cooling and produces beads with controlled porosity and size due to its low viscosity. Such a process is disclosed, for example, in WO2020221762. However, in recent years, growing environmental concerns have led the industry to develop production methods that avoid the use of organic solvents and to replace them partially or completely with natural or plant alternatives. Furthermore, the use of organic solvents such as toluene also poses other problems, such as explosion hazards and occupational health and safety concerns.

[0005] Agar consists of agarose and agaropectin, usually in a ratio of 90:10 to 70:30. Both agarose and agaropectin are polysaccharides consisting of alternating anhydrogalactose and galactose subunits, i.e., their polysaccharide backbones are the same. Agaropectin is heavily sulfated and therefore negatively charged. It is also methylated, meaning that it contains methoxy groups. Agarose is essentially uncharged and has no sulfate groups. Both agar, particularly desulfated agar, and agarose were originally proposed as starting materials for the production of cross-linked separating gels. See, for example, U.S. Pat. No. 3,959,251 (Porath et al.). However, in the last few years, people have focused more on agarose than on agar. This is probably due to the high content of sulfate groups (contained in agaropectin) in agar and the problems in removing the sulfate groups without adversely affecting the quality of the agar base.

[0006] It has been shown previously that vegetable oils can be used to replace organic solvents as the continuous phase of water-in-oil emulsions when producing agarose beads, e.g., NICOLAS IONNIDIS et al., Journal of Colloid and Interface Science 367 (2012), and CHEN et al., Journal of Separation Science, vol 40, 22 / 17. However, all these results were generated at laboratory scale and therefore in very small quantities.

[0007] Controlling the cooling of emulsions with vegetable oil as the continuous phase is more difficult due to the significantly higher viscosity of vegetable oil compared to organic solvents. When emulsifying on a small scale, it is less of an issue due to the smaller volumes to be handled, and therefore jacket cooling of the reactor is a good option to cool emulsions with a high viscosity continuous phase. However, this is not an option for industrial scale production, as the combination of jacket cooling and the higher viscosity of vegetable oils results in slow and uncontrollable cooling, which in turn results in loss of shape, size and porosity characteristics of the beads. Slow cooling results in a relatively high porosity; rapid cooling results in smaller pores.

[0008] It would be advantageous to produce agar or agarose beads by a method that does not utilize organic solvents such as toluene in an industrially viable process. Additionally, it would be advantageous to produce agar or agarose beads with a controlled size and / or porosity distribution. Summary of the Invention

[0009] It is an object of the present invention to mitigate or obviate one or more of the above disadvantages by providing an improved method for producing agar or agarose beads.

[0010] Another object of the present invention is to provide a method for the preparation of agar or agarose beads using a water-in-oil (W / O) emulsion with natural or vegetable oil as the continuous (oil) phase, thereby achieving a method that eliminates or reduces the use of low viscosity organic solvents.

[0011] Another object of the present invention is to provide a method for producing agar or agarose beads, which includes a cooling step, suitable for use in industrial scale production.

[0012] Another object of the present invention is to provide a method for producing agar or agarose beads that results in agar or agarose beads with a controlled size distribution and shape.

[0013] It is another object of the present invention to provide a method for producing agar or agarose beads that results in agar or agarose beads with controlled porosity.

[0014] Another object of the present invention is to provide a method for producing agar or agarose beads that reduces or eliminates oil content in the beads.

[0015] Another object of the present invention is to provide agar or agarose beads suitable for use as a chromatography resin.

[0016] In one general embodiment of the invention, a method for producing agar or agarose beads is provided, the method comprising: (i) providing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature above the gelling temperature of the aqueous solution; (ii) providing an oil phase comprising a natural or vegetable oil at a temperature higher than the gelling temperature of the aqueous solution provided in step (i); (iii) mixing the aqueous phase provided in step (i) with the oil phase obtained in step (ii) in a reactor and adding an emulsifier; (iv) emulsifying the mixture obtained in step (iii), preferably by agitating the mixture, thereby producing an emulsion; (v) performing staged cooling of the emulsion obtained in step (iv), comprising a first cooling step for cooling the emulsion to a temperature 0.1-30° C. higher than the gelling temperature of the aqueous solution obtained in step (i), followed by a second cooling step for cooling the emulsion to a temperature lower than the gelling temperature of the aqueous solution provided in step (i) by emptying the emulsion from the reactor and passing the emulsion through a heat exchanger; (vi) recovering the agar or agarose beads from the emulsion; Includes.

[0017] The method according to the invention achieves a more controlled particle size distribution and porosity. Without being bound to any particular theory, it is believed that by cooling the emulsion stepwise and initially bringing the emulsion to a temperature close to, but higher than, the gelling temperature of the aqueous solution of agar or agarose, the viscosity difference between the start of cooling and the gelling point, i.e., the temperature at which the "viscous" beads turn into gelled (i.e., solid) beads, is reduced. The temperature gradient is easier to control by reducing the temperature and viscosity difference between the start temperature at which the beads are still in a viscous form and a temperature below the gelling temperature, i.e., the temperature at which the beads are in a gelled (i.e., solid) form. This allows for better control of temperature-sensitive parameters such as size distribution and porosity, which are greatly affected by the cooling rate. When preparing emulsions with a continuous phase that exhibits higher viscosity, the control of cooling is very important and therefore the temperature control during cooling is more difficult compared to conventional organic solvents.

[0018] Final cooling of the emulsion through a heat exchanger achieves rapid cooling with a controlled temperature gradient, allowing control of the gelation temperature profile and thus the size distribution and porosity of the resulting beads. Furthermore, by achieving rapid and controlled cooling, the method can be used for industrial-scale production, even when a high viscosity continuous phase is used for water-in-oil (W / O) emulsions.

[0019] The aqueous phase, including an aqueous solution of agar or agarose, can be native agar, native agarose, or a derivative of agar or agarose, for example, as described in WO2008136742 and U.S. Pat. No. 6,602,990, which are incorporated by reference in their entireties.

[0020] The gelling temperature of an agar or agarose solution is usually above 40°C. However, slight variations from this value occur depending on the amount and purity of agar present in the solution. However, those skilled in the art are expected to be aware of these and understand that such variations exist. In one embodiment of the present invention, the aqueous phase containing the aqueous solution of agar or agarose is prepared at a temperature above 40°C, preferably between 40 and 99°C, preferably between 40.1 and 99.9°C, even more preferably between 41°C and 95°C.

[0021] In one embodiment of the present invention, the oil phase is provided at a temperature above 40°C, preferably between 40 and 99°C, preferably between 40.1 and 99.9°C, even more preferably between 41°C and 95°C.

[0022] In one embodiment, the emulsifier is added to the mixed solution after both the water phase and the oil phase are mixed. Preferably, the emulsifier is added after the water phase is added to the oil phase.

[0023] In one embodiment, step (iv) of emulsifying the mixture may be carried out by any conventional emulsification method known to one of skill in the art.

[0024] The first cooling step for cooling the emulsion obtained in step (iv) cools the emulsion to a temperature 0.1 to 30° C. higher than the gelation temperature of the aqueous solution obtained in step (i). In one embodiment, the first cooling step cools the emulsion to a temperature 0.5 to 15° C. higher than the gelation temperature of the aqueous solution prepared in step (i), preferably to a temperature 0.5 to 10° C. higher than the gelation temperature of the aqueous solution prepared in step (i), more preferably to a temperature 0.5 to 5° C. higher than the gelation temperature of the aqueous solution prepared in step (i). In one embodiment, the first cooling step cools the emulsion to a temperature of 40.5 to 49° C., preferably to a temperature of 41 to 45° C.

[0025] By using stepwise cooling in accordance with the present invention, it is possible to utilize a more viscous oil phase compared to the organic solvent because the cooling is accomplished in a fast, controlled and convenient manner.

[0026] Recovery of the agar or agarose beads from the formed emulsion can be by any conventional means known to those skilled in the art. In one embodiment, the beads are recovered by settling them in the presence of excess water, and optionally a surfactant to separate the beads from the aqueous phase.

[0027] In one embodiment of the present invention, step (iii) is carried out by adding the aqueous solution from step (i) to the oil phase prepared in step (ii) in a reactor, preferably by pouring the aqueous solution from step (i) into the reactor containing the oil phase from step (ii). The inventors have surprisingly discovered that the addition method has a significant impact on the formation of beads in the emulsion. Traditionally, when emulsifying using a low viscosity solvent such as toluene, toluene is added to the aqueous phase. However, when this addition method is used for high viscosity oils such as vegetable oils, the subsequently formed beads will be contaminated with oil, compromising their quality. The inventors have solved this problem by adding the aqueous phase instead of the oil phase. Preferably, the addition of the aqueous phase to the oil phase is carried out in a controlled manner, for example by injection or dropwise, to ensure a more uniform distribution and avoid agglomeration. It has also been shown that the temperature of the aqueous phase when added to the oil phase affects the final properties of the beads. Preferably, the aqueous phase is added at a temperature higher than 70°C, preferably higher than 80°C, preferably higher than 90°C, even more preferably higher than 95°C.

[0028] In one embodiment of the invention, steps (iii) and (iv) are carried out simultaneously. In one embodiment of the invention, step (iv) begins after the aqueous phase has been added to the oil phase.

[0029] In one embodiment of the present invention, the first cooling step is performed by cooling the emulsion in the reactor to a temperature of 0.1-20°C above 40°C, preferably 1-10°C above 40°C, more preferably 1-5°C above 40°C. As mentioned above, cooling the emulsion to a temperature closer to the gelling temperature of the aqueous agar or agarose solution (i.e., the aqueous phase) reduces the viscosity gap between the start of cooling and the gelling temperature of the beads. This allows for better control of the temperature gradient and parameters such as bead porosity and size distribution.

[0030] In one embodiment of the invention, the second cooling step cools the emulsion to a temperature below 30° C., preferably below 25° C. Beads are obtained by cooling to a temperature below the gelling temperature of the aqueous solution (i.e., the aqueous phase) of the agar or agarose solution.

[0031] Natural oils can be oils obtained from various parts of oil-containing plants, such as oils from seeds, fruits, leaves, flowers, stems, bark or roots.In one embodiment of the present invention, vegetable oils are selected from rapeseed oil, corn oil, sunflower oil, peanut oil or other plant-based oils.Preferably, vegetable oil is rapeseed oil.It has been shown that rapeseed oil shows optimal viscosity characteristics to ensure controlled cooling of emulsion.

[0032] In one embodiment of the present invention, the stirring in step (iv) is performed by an overhead mixer, preferably at 1000-2000 rpm, even more preferably at 1250-1750 rpm. Traditionally, when producing agar or agarose beads, high shear mixers are used. However, when using oils with higher viscosity than traditional organic water immiscible solvents, high shear mixing results in the oil being mixed into the beads. Without being bound to any particular theory, it is believed that the increase in viscosity increases the mechanical impact on the beads during mixing compared to the same process in a low viscosity oil phase. However, it has been shown that with a high viscosity oil phase, the use of high speed conventional mixers allows for good particle distribution of the beads. The speed of the mixer (revolutions per minute or rpm) has been shown to affect the size distribution of the beads. As the speed increases, the shear effect becomes stronger and the Dv50 value of the beads decreases. Dv50 means that 50% of the product weight is below a particular micron size. Also, high speeds can force the continuous phase (oil phase) into the formed emulsion globules, creating emulsions within emulsions and compromising the properties of the resulting beads. Too low a speed will not produce an emulsion.

[0033] In one embodiment of the invention, the second cooling step comprises passing the emulsion through a series of heat exchangers. The use of a series of heat exchangers allows providing different cooling settings and thereby controlling the cooling gradient in different ways.

[0034] In one embodiment of the invention, the second cooling step comprises passing the emulsion through a 100-700 kW heat exchanger, preferably a 600-700 kW heat exchanger. Preferably, the temperature of the water passing through the heat exchanger is between 5 and 20°C, more preferably between 7 and 15°C.

[0035] In one embodiment of the present invention, the volume ratio of the aqueous phase to the oil phase is 1:9 to 1:1, preferably 1:4 to 1:1, and preferably 2:5 to 5:8. If the volume of the aqueous phase is large relative to the volume of the oil phase, the Dv50 value becomes high.

[0036] In one embodiment of the present invention, the emulsifier is a non-ionic surfactant, preferably the emulsifier is a sorbitan ester. In one embodiment, the emulsifier is selected from the SPAN family, preferably the emulsifier is selected from Span™ 80, Span™ 85, or a mixture thereof. The type of emulsifier influences the size of the beads and also the shape of the beads. Inappropriate emulsifier selection can cause bead clumping and oil contamination. Emulsifiers reduce the interfacial tension between the oil and water phases, promoting their division into smaller droplets and stabilizing them. Insufficient interaction usually results in malformation. To obtain spherical agar or agarose beads when using a high viscosity oil phase, emulsifiers selected from sorbitan esters, i.e. SPAN, are shown to minimize the generation of deformed beads and maintain a good size distribution.

[0037] Emulsifiers are characterized by their HLB value, which is an indication of the solubilizing properties of the emulsifier and indicates the type of emulsion (O / W or W / O) for which the emulsifier is best suited. For water-in-oil (W / O) emulsions, a low HLB is preferred, as this means a higher solubility in the continuous (oil) phase, contributing to a larger proportion of beads within the controlled size range compared to emulsifiers with higher HLB values.

[0038] In one embodiment of the invention, the mixture obtained in step (iii) comprises the emulsifier in an amount of 10-20 g / L oil phase, preferably 12.5-17.5 g / L oil phase.

[0039] In one embodiment of the present invention, step (iv) is carried out at 60-95°C.

[0040] In one embodiment of the present invention, the aqueous solution of agar or agarose comprises 1-9 wt% agar or agarose, preferably 3-8 wt% agar or agarose, and even more preferably about 7 wt% agar or agarose. The agar or agarose content of the aqueous solution determines the viscosity of the aqueous phase of the emulsion. Viscosity is important in determining how well the phases are mixed and therefore the size of the resulting beads in the emulsion. Generally, an increased agar content produces larger and denser beads with lower porosity values. Furthermore, a higher agar content is believed to reduce the oil content. The lower the agar content, the less energy input is required to shear the mixture and emulsify it.

[0041] In one embodiment of the present invention, the volume ratio of the aqueous phase to the oil phase is 2:8.

[0042] In one embodiment of the present invention, the aqueous phase may further comprise at least one salt. The salt increases the gelling temperature while decreasing the viscosity. In one embodiment of the present invention, the aqueous phase may further comprise an acid. A low pH reduces the viscosity of the aqueous phase, which affects the strength of the gel. In one embodiment of the present invention, the oil phase may further comprise an antifoaming agent.

[0043] In a second general embodiment of the invention, there is provided an agar or agarose bead obtained by a method according to any of the preceding embodiments. d As measured by thyroglobulin, it exhibits a porosity of 0.20-0.35, and more than 50% of the beads exhibit a size between 30-75 μm.

[0044] In one embodiment of the invention, more than 55% of the beads exhibit a size between 30 and 75 μm.

[0045] In gel filtration, the distribution of a particular compound between the inner and outer mobile phases is a function of its molecular size and is known as the distribution coefficient (K d ) for larger molecules that are usually excluded from gel beads. d The value of K is 0. Certain molecules smaller than the pore size of the gel beads enter the pores of the gel matrix and therefore d The value is 1. For medium-sized molecules, K d The value is between 0 and 1. d This type of variation in value allows for the separation of molecules in a narrow molecular size range.

[0046] The invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0047] [Figure 1] Showing 10x microscopic images of agarose beads produced by emulsions of agar solution in rapeseed oil using different rates of addition of agar solution to the oil phase, Figure 1 is divided into left and right halves, with each half separated by a dashed line. [Diagram 2] Showing 10x microscopic images of agarose beads produced by emulsion of agar solution in rapeseed oil using various loading techniques, Figure 2 is divided into left and right halves, each half separated by a dashed line. [Diagram 3]Showing 10x microscopic images of agarose beads produced by emulsions of agar solution in rapeseed oil and toluene, respectively, Figure 3 is divided into left and right halves, each half separated by a dashed line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] As used herein, "wt %" refers to the weight percent of a referenced component relative to the total weight of the referenced compound or composition.

[0049] As used herein, "approximately" should be interpreted as being as precise as the method used to measure the referenced value.

[0050] The present invention relates to a method for producing agar or agarose beads suitable for use as a chromatography resin. The method utilizes a water-in-oil (W / O) emulsion containing a natural or vegetable oil as the oil phase (continuous phase). When the resulting emulsion is cooled by the stepwise cooling of the present invention, gelled (solidified) beads are formed. Stepwise cooling according to the present invention allows the method to be used in industrial scale processes since cooling is performed in a fast, convenient and controlled manner.

[0051] As mentioned before, when replacing high viscosity vegetable oils with low viscosity organic solvents such as toluene, some problems arise due to the different properties of the oil phase. As the viscosity increases, the temperature control of the cooling becomes more difficult. Without a controlled temperature gradient, it becomes more difficult to control parameters such as the size distribution and pore size of the resulting beads. The increased viscosity also raises the question of how to combine the different phases.

[0052] (Method of combining oil and water phases) As mentioned before, when the viscosity of the continuous phase (oil phase) is high, it is shown that how the dispersed phase (water phase) is added to the system is very important. When a low viscosity continuous phase such as toluene is used, no difference is shown in the final product when the continuous phase is added to the agar solution and vice versa. In this case, the rate of addition also does not seem to affect the results. In the case of a high viscosity continuous phase (rapeseed oil), however, if the agar is added too quickly (see Figure 1) or if oil is added to the agar instead ("reverse" "emulsion") (see Figure 2), there is an oil inclusion in the final product.

[0053] FIG. 1 shows a 10x microscope image of agarose beads produced by emulsion of agar solution in rapeseed oil. The beads are produced using a standard emulsion consisting of 4:1 rapeseed oil and agar solution (7%) and 15 g / L Span™ 85 oil phase, stirred with an overhead stirrer (1500 rpm) at 90° C. Staged cooling is performed, with a first cooling step carried out to cool the emulsion to 40° C. in the reactor, and then a second cooling step through a 115 kW heat exchanger. The left half of FIG. 1 shows beads produced by slow addition of agar solution to the oil phase. The right half of FIG. 1 shows beads produced by rapid addition of agar solution to the oil. As can be seen in FIG. 1, rapid addition results in oil contamination of the beads (see the right half of FIG. 1).

[0054] FIG. 2 shows a 10x microscope image of agarose beads produced by emulsion of agar solution in rapeseed oil. The beads are produced using a standard emulsion consisting of 4:1 rapeseed oil to agar solution (7%) and 15 g / L Span™ 85 oil phase, stirred with an overhead stirrer (1500 rpm) at 90°C. A stepwise cooling is carried out, with a first cooling step carried out to cool the emulsion to 40°C in the reactor, and then a second cooling step through a 115 kW heat exchanger. The left half of FIG. 2 shows beads produced by adding oil to the agar solution. The right half of FIG. 2 shows beads produced by rapidly adding agar solution to oil. As can be seen from FIG. 2, when oil is added to the agar solution, the oil is mixed into the beads (see the left half of FIG. 2). This phenomenon is not seen when agar solution is added instead of oil.

[0055] The agar should also preferably be added warm (approximately 95° C.) to avoid localized gelling.

[0056] (Mixer type and RPM (revolutions per minute)) Traditionally, when producing agar or agarose beads, high shear mixers are used to effectively create small droplets in the desired size range. However, this method cannot be used with emulsions that contain a high viscosity continuous phase due to the possibility of oil contamination. Without being bound to a particular theory, it is believed that the increased viscosity increases the mechanical impact on the beads during mixing compared to the same process with a low viscosity oil phase. However, it has been shown that a high viscosity oil phase allows for good particle distribution of the beads by using high speed conventional mixers.

[0057] As can be seen from Figure 3, the mixing speed has a significant effect on the distribution of beads. Figure 3 shows 10x microscope images of agarose beads produced by emulsion of agar solution in rapeseed oil and toluene, respectively. The beads are produced using an emulsion consisting of 4:1 oil phase and 20% agar solution (7%) and 15 g / L Span™ 85 oil phase, stirred at 8000 rpm and 90°C in a high shear mixer. A staged cooling is carried out, with a first cooling step carried out to cool the emulsion to 40°C in the reactor, and then a second cooling step through a 115 kW heat exchanger. The left half of Figure 3 shows beads produced in toluene. The right half of Figure 3 shows pearls in rapeseed oil. As can be seen, the quality of the beads is compromised when using a high shear mixer with rapeseed oil (see the right half of Figure 3). The beads produced using the lower rpm shown in Figures 1 and 2 show less oil inclusion and an overall better dispersion.

[0058] (Ratio of dispersed phase (water phase) to continuous phase (oil phase)) As mentioned before, the ratio of the dispersed phase (water phase) to the continuous phase has a large influence on the size distribution and pore size of the beads. Table 1 shows the results of an experimental series based on eight different experiments, where four different parameters and their influence on the Dv50 and the percentage of beads between 30 and 75 μm are investigated. The parameter with the greatest influence in this series of experiments is the ratio of the dispersed phase to the continuous phase (AgOil).

[0059] Dv50 means that 50% of the product's weight is below a particular micron size.

[0060] [Table 1]

[0061] (emulsifier) In water-in-oil emulsions, a low HLB is preferred since it means a higher solubility in the continuous (non-polar) phase, which contributes to a larger percentage of beads that are in the range of 30-75 μm compared to emulsifiers from the same chemical family but with higher HLB values. Two different emulsifiers from the SPAN family with different HLB values ​​were investigated. The results are shown in Table 2. Table 2 shows the difference in particle distribution of beads emulsified with Span™ 80 and Span™ 85. The beads are produced using a standard emulsion consisting of a 4:1 rapeseed oil to agar solution (5.3%) and 15 g / L Span™ 85 oil phase, stirred with an overhead stirrer (1500 rpm) at 90 °C. A staged cooling is performed, with a first cooling step carried out to cool the emulsion to 55 °C in the reactor, and then a second cooling step via a 115 kW heat exchanger.

[0062] As shown in Table 2, the lower the HLB value, the more beads there are within a particular size range and the narrower the overall size distribution.

[0063] [Table 2]

[0064] (First cooling step) As mentioned before, the size control is further improved by using staged cooling, where the first cooling step cools the emulsion to a temperature close to the gelling temperature of the agar or agarose solution. Table 3 shows the effect of the first cooling step on the size of the beads. The table shows the difference in particle distribution of beads cooled from different temperatures, i.e. different first cooling step temperatures. The beads are produced using a standard emulsion consisting of 4:1 rapeseed oil to agar solution (7%) and 15 g / L Span™ 85 oil phase, stirred with an overhead stirrer (1500 rpm) at 90°C. Cooling is first performed in a reactor to the specified temperature (first cooling step) and then through a 115 kW heat exchanger (second cooling step).

[0065] As can be seen, increasing the starting temperature of the second cooling step (increasing the temperature of the first cooling step) results in fewer beads being achieved within the desired size specification. The conclusion drawn from this experiment is that the temperature of the first cooling step significantly affects the size distribution of the beads.

[0066] [Table 3]

[0067] (cooling method) Different cooling techniques are evaluated: cooling in the reactor, cooling in a cooling vessel and cooling using a heat exchanger. The results are shown in Table 4. The beads are produced using a standard emulsion consisting of a 4:1 rapeseed oil to agar solution (4.7%) and 15 g / L Span™ 85 oil phase at 90°C and stirred with an overhead stirrer (1500 rpm).

[0068] Cooling in the reactor was achieved by running cold tap water through the reactor jacket while stirring the hot emulsion mixture. This method allowed the emulsion mixture to be cooled uniformly over a long period of time. Cooling in this manner reduced the K d values, resulting in porous beads that tend to be slightly softer (see Table 4). However, this type of cooling leads to the beads hardening and the formation of permanent agglomerates.

[0069] Cooling in the cooling vessel was achieved by pouring the hot emulsion liquid onto a cold cooling medium, which resulted in instant cooling to the final temperature. This rapid cooling reduces the porosity of the beads, which is known as the K d This is reflected in a decrease in the value of 0.1%. However, additional refrigerant is required in the form of a continuous phase and an open vessel, which is not required in the other methods. As a result, the method is not optimal for industrially large-scale production.

[0070] Cooling by heat exchanger was achieved by passing the hot emulsion mixture through a cooled heat exchanger, which allowed for the cooling of beads with similar porosity characteristics as those in a cooling vessel, i.e., a relatively low K d A bead with a value is obtained.

[0071] As can be seen in Table 4, cooling with a heat exchanger increased the amount of beads within the desired size specification and narrowed the size distribution compared to other cooling techniques.

[0072] [Table 4]

[0073] (Effect of staged cooling on porosity) In the next example, the effect of stepwise cooling on the porosity of the formed beads was investigated. As previously mentioned, stepwise cooling of the emulsion allows for an easier control of the cooling temperature gradient by first bringing the emulsion close to, but still above, the gelling temperature of the aqueous solution of agar or agarose, and then cooling the emulsion below the gelling temperature. The porosity of the formed beads is strongly influenced by the cooling rate.

[0074] An agar solution containing 7% agar in water is produced. This solution is heated to a temperature of 94°C and poured with stirring into an oil phase containing rapeseed oil and Span 85. The combined agar solution and oil phase are stirred at 94°C and 980 RPM. The resulting emulsion consists of a 4:1 rapeseed oil to agar solution (7%) oil and a 15g / L Span™ 85 oil phase.

[0075] A first cooling step is performed to cool the emulsion in the reactor to 43 °C. The cooled emulsion is transferred to a 660 kW heat exchanger connected to chilled tap water at 12 °C. The emulsion is cooled to 14-18 °C and the formed agar beads are collected.

[0076] The resulting beads K dThe porosity measured with thyroglobulin is shown in Table 5.

[0077] [Table 5]

[0078] As you can see, K d The thyroglobulin values ​​are significantly lower compared to the beads shown in Table 4 that were produced using a single cooling step and similar agar concentrations. Thus, this method results in agar beads that exhibit a porosity comparable to available commercial agar beads formed using toluene as the oil phase.

Claims

1. 1. A method for producing agar or agarose beads suitable for use as a chromatography resin, comprising the steps of: (i) providing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature above the gelling temperature of said aqueous solution; (ii) providing an oil phase comprising a natural or vegetable oil at a temperature higher than the gelling temperature of the aqueous solution provided in step (i); (iii) mixing the aqueous phase provided in step (i) with the oil phase obtained in step (ii) in a reactor and adding an emulsifier; (iv) emulsifying the mixture obtained in step (iii), preferably by agitating said mixture, thereby producing an emulsion; (v) performing staged cooling of the emulsion obtained in step (iv), comprising a first cooling step for cooling the emulsion to a temperature 0.1-30° C. higher than the gelling temperature of the aqueous solution obtained in step (i), followed by a second cooling step for cooling the emulsion to a temperature lower than the gelling temperature of the aqueous solution provided in step (i) by emptying the emulsion from the reactor and passing the emulsion through a heat exchanger; (vi) recovering the agar or agarose beads from the emulsion; A manufacturing method comprising:

2. 2. The process according to claim 1, wherein step (iii) is carried out by adding said aqueous solution from step (i) to the oil phase provided in step (ii) in said reactor, preferably by pouring said aqueous solution from step (i) into said reactor containing said oil phase from step (ii).

3. 2. The method of claim 1, wherein steps (iii) and (iv) are performed simultaneously.

4. 2. The method according to claim 1, wherein the first cooling step is carried out by cooling the emulsion in the reactor to a temperature of 0,1 to 20°C above 40°C, preferably to a temperature of 1 to 10°C above 40°C, preferably to a temperature of 1 to 5°C above 40°C.

5. 2. The method of claim 1, wherein the second cooling step cools the emulsion to a temperature below 30°C, preferably below 25°C.

6. 2. The method of claim 1, wherein the vegetable oil is selected from rapeseed oil, corn oil, sunflower oil, peanut oil, or other vegetable-based oil.

7. The process according to claim 1, wherein the stirring in step (iv) is carried out by an overhead mixer, preferably at 1000-2000 rpm, even more preferably at 1250-1750 rpm.

8. 10. The method of claim 1, wherein the second cooling step comprises passing the emulsion through a series of heat exchangers.

9. The method of claim 1, wherein the second cooling step comprises passing the emulsion through a 100-700 kW heat exchanger, preferably a 600-700 kW heat exchanger.

10. The process according to claim 1, wherein the volume ratio of the aqueous phase to the oily phase is from 1:9 to 1:1, preferably from 1:4 to 1:1, preferably from 2:5 to 5:

8.

11. The method of claim 1 , wherein the emulsifier is a non-ionic surfactant, preferably the emulsifier is a sorbitan ester.

12. 2. The method of claim 1, wherein the mixture obtained in step (iii) comprises emulsifier in an amount of 10 to 20 g / L oil phase, preferably 12.5 to 17.5 g / L oil phase.

13. The method of claim 1, wherein step (iv) is carried out at 60 to 95° C.

14. 2. The method of claim 1, wherein the aqueous solution of agar or agarose comprises 1-9 wt% agar or agarose, preferably 3-8 wt% agar or agarose, even more preferably about 7 wt% agar or agarose.

15. The beads are K ｄ Agar or agarose beads obtained by the method according to any one of claims 1 to 14, exhibiting a porosity, measured by thyroglobulin, between 0.20 and 0.35, with more than 50% of the beads exhibiting a size between 30 and 75 μm.

16. Agar or agarose beads according to claim 15, in which more than 55% of the beads exhibit a size between 30 and 75 μm.

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