Method for making agar or agarose beads

Abstract

The present invention discloses a method for making agar or agarose beads, comprising the steps of: a) providing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature of 40-100°C; b) providing an oil phase comprising a water-immiscible solvent and an emulsifier at a temperature of 40-100°C; c) emulsifying the aqueous phase in the oil phase to form a water-in-oil emulsion; d) cooling the water-in-oil emulsion to a temperature below the gelling temperature of the agar or agarose to form a dispersion of solidified agar or agarose beads; and e) recovering the agar or agarose beads from the dispersion, wherein the emulsifier comprises a phosphate ester of an alkoxylated fatty alcohol.

Description

Technical Field

[0001] The present invention relates to agar / agarose beads, more particularly to a method for making agar or agarose beads. The present invention also relates to an emulsifier suitable for use in the method. Background Art

[0002] For decades, agarose beads have been used as stationary phases in the chromatographic separation of proteins and other biomacromolecules. They are usually prepared by reversed suspension gelation, wherein a hot aqueous solution of agarose or agar is emulsified in a hot oil phase to form a water-in-oil (W / O) emulsion. The emulsion is then cooled to below the gelling temperature of agarose / agar to produce gel beads, which can then be recovered and used for separation purposes. These methods are described in, for example, S Hjertén: Biochim Biophys Acta 79 (2), 393-398 (1964), WO1989011493A1 and US20180171484, which are incorporated herein by reference in their entirety. A variant is described in US20100084345, in which agarose beads are converted into agarose beads by hydrolysis of sulfate groups after gelation, and the document is also incorporated by reference in its entirety.

[0003] In this method, the use of an emulsifier to stabilize the W / O emulsion is essential. The emulsifier is also important for the size distribution of the resulting beads and their shape. In addition, the emulsifier should be easily removed from the beads by washing, and it should be environmentally friendly and not produce any toxic leachables when the beads are used to manufacture drugs.

[0004] Previously disclosed emulsifiers lack several of these aspects and, therefore, additional emulsifiers are needed. Summary of the invention

[0005] One aspect of the present invention is to provide a method for making agar or agarose beads. This is achieved by a method comprising the following steps:

[0006] a) providing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature of 40-100° C.;

[0007] b) providing an oil phase comprising a water-immiscible solvent and an emulsifier at a temperature of 40-100° C.;

[0008] c) emulsifying the aqueous phase in the oil phase to form a water-in-oil (W / O) emulsion;

[0009] d) cooling the W / O emulsion to a temperature below the gelling temperature of agar or agarose to form a dispersion of solidified agar or agarose beads; and

[0010] e) recovering agar or agarose beads from the dispersion,

[0011] The emulsifier comprises a phosphoric acid ester of an alkoxylated fatty alcohol.

[0012] One advantage is that aggregation of the beads in step d) is prevented, resulting in well-dispersed beads of high sphericity. Other advantages are that the emulsifier is water-soluble, easy to remove by water washing, and it does not contain endocrine disruptors such as alkylphenol derivatives.

[0013] Another aspect of the present invention is to provide agar or agarose beads obtainable by the above method.

[0014] Further suitable embodiments of the invention are described in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown are examples of well-dispersed spherical agarose beads formed from a well-stabilized W / O agarose emulsion.

[0016] Figure 2 Shown are examples of agarose beads with aggregates (indicated by arrows) that may form during the cooling of a W / O agarose emulsion.

[0017] Figure 3 Shown are examples of agarose beads with non-spherical, partially agglomerated beads (indicated by arrows).

[0018] Figure 4 An example of agarose beads is shown with spherical inclusions in the beads (indicated by arrows) resulting from O / W / O double emulsion formation. DETAILED DESCRIPTION

[0019] In one aspect, the present invention discloses a method for making agar or agarose beads. The method comprises the following steps:

[0020] a) providing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature of 40-100° C. The aqueous phase may, for example, comprise 1-8% by weight of agar or agarose, such as 2-7% by weight of agar or agarose, such as about 2% by weight, about 4% by weight, or about 6% by weight. The aqueous phase may further comprise one or more buffer components, such as a weak base. The agar or agarose may be natural agar, natural agarose, or a derivative of agar or agarose, such as allyl agarose or hydroxyethyl agarose, as further described in US6602990 and US7396467, which are incorporated herein by reference in their entirety;

[0021] b) providing an oil phase comprising a water-immiscible solvent and an emulsifier at a temperature of 40-100°C (suitably at a temperature below the boiling point of the solvent). The water-immiscible solvent may be, for example, a hydrocarbon, an ester or a ketone. In order to facilitate efficient solvent recovery, the solvent may have a boiling point in the range of about 90-170°C at atmospheric pressure, for example 90-150°C or 100-120°C. It may be, for example, toluene (boiling point 111°C) or xylene (boiling point about 140°C: m-xylene 139°C and o-xylene 144°C). Alternatively, it may be a cyclic ketone, such as 2-methylcyclohexanone (boiling point 162-163°C). Higher boiling point solvents, such as mineral oil or vegetable oil, may also be used, although the solvent may be more difficult to recover. The properties of the emulsifier are further described below. The emulsifier may be a single emulsifier, or the oil phase may contain a mixture of several emulsifiers. The concentration of emulsifier in the oil phase (or the total emulsifier concentration) may be, for example, 0.01-2% by weight, such as 0.015-1% by weight. Suitably, none of the emulsifiers comprises an alkylphenol or an alkylphenol derivative. The oil phase may, for example, comprise less than 0.1% by weight, such as less than 0.01 or less than 0.001% by weight of an alkylphenol or an alkylphenol derivative. It may even be free of alkylphenol and alkylphenol derivatives;

[0022] c) emulsifying the aqueous phase in the oil phase to form a water-in-oil (W / O) emulsion. Emulsification may include mixing the aqueous phase and the oil phase in a stirred vessel to form the W / O emulsion. This step may further include passing the W / O emulsion through a rotor-stator mixer, a static mixer, or a porous membrane to reduce the droplet size of the W / O emulsion. In another way of forming the emulsion, the aqueous phase may enter the oil phase through a porous membrane or a sieve plate to form the W / O emulsion;

[0023] d) cooling the W / O emulsion to a temperature below the gelling temperature of agar or agarose to form a dispersion of solidified agar or agarose beads. This step can be performed by gradually cooling the emulsion in a stirred vessel, or it can be performed in a continuous mode by passing the W / O emulsion through a conduit having a longitudinally decreasing temperature gradient;

[0024] e) recovering the agar or agarose beads from the dispersion. The recovery may comprise adding water or an aqueous solution to the dispersion obtained in step d), decanting the oil phase, and recovering the agar or agarose beads as a precipitate. The beads may be further washed with water or an aqueous solution to remove residual emulsifier and / or other substances. Washing with an organic solvent may also be used to remove emulsifier residues and other leachables.

[0025] After step e), the beads may be cross-linked in step f) by adding a cross-linking agent such as epichlorohydrin. They may be further functionalized with ligands in step g), wherein the ligands are covalently coupled using methods known to those skilled in the art. Step g) may suitably be performed after step f), although it is also possible to couple the ligands to non-cross-linked beads.

[0026] The beads prepared by this method may be used in chromatographic separation methods or batch adsorption methods.They may for example have a diameter in the range of 5-500 μm (expressed as volume weighted median diameter d50, v), for example 10-350 μm or 30-120 μm.

[0027] The above-mentioned emulsifier includes the phosphoric acid ester of alkoxylated fatty alcohol. Generally, it can contain the mixture of the phosphoric acid monoester and phosphoric acid diester of alkoxylated fatty alcohol. Fatty alcohol can contain one or more C 10 -C 20 The linear or branched, primary or secondary alkanol or alkenol and / or ethoxylated fatty alcohol may have structure I

[0028] R1 – O – (R2-O) n -H (I)

[0029] in:

[0030] R1 is a saturated or unsaturated, linear or branched aliphatic C 10 -C 20 Hydrocarbons, such as saturated or unsaturated linear aliphatic C 10 -C 18 hydrocarbon,

[0031] R2 is -CH2-CH2-, or a mixture of -CH2-CH2- and -CH2(CH3)-CH2-, and

[0032] n is 2-20, such as 2-10 or 2-5.

[0033] This structure describes an ethoxylated or mixed ethoxylated / propoxylated fatty alcohol with an average degree of alkoxylation of n. The alkoxylated fatty alcohol is then converted to a phosphate ester of structures II and III, where II is a phosphate monoester and III is a phosphate diester:

[0034] R1-O- (R2-O) n -P (O) (OH) -OH (II)

[0035] R1-O- (R2-O) n -P (O) (OH) - (O-R2) n -O-R1 (III)

[0036] in:

[0037] R1 is a saturated or unsaturated, linear or branched aliphatic C 10 -C 20 Hydrocarbons, such as saturated or unsaturated linear aliphatic C 10 -C 18 hydrocarbon,

[0038] R2 is -CH2-CH2-, or a mixture of -CH2-CH2- and -CH2(CH3)-CH2-, and

[0039] n is 2-20, such as 2-10 or 2-5.

[0040] Both the phosphoric acid monoester II and the diester III are acidic compounds with dissociable acidic hydrogens. Therefore, the phosphoric acid ester emulsifier of the present invention can be provided in acid form or in neutralized form (e.g., sodium salt or potassium salt or ammonium salt). If an emulsifier in acid form is used, a base can be appropriately added to the aqueous phase to adjust the pH to near neutrality, because agar and agarose are sensitive to degradation under acidic conditions. In addition to the monoester II and the diester III, the emulsifier can also include the corresponding phosphoric acid triester of the alkoxylated fatty alcohol and the free, unesterified alkoxylated fatty alcohol. Both compounds are non-acidic and are usually present in small amounts, such as <25% by weight or less than 15% by weight of the emulsifier.

[0041] In particular, the emulsifier may contain C 10 -C 16 A mixture of monoesters and diesters of phosphoric acid of mixed ethylene oxide + propylene oxide adducts of alkanols. Such products can be Lubrhophos TM Alternatively, the emulsifier may comprise a mixture of phosphoric acid monoesters and phosphoric acid diesters of ethoxylated oleyl alcohol with n=3 (average number of ethylene glycol units per oleyl alcohol). Such products are generally known by the INCI (International Nomenclature of Cosmetic Ingredients) name oleth-3-phosphate and may be marketed under the trade name Crodafos TM O3A (Croda) is commercially available. Example

[0042] Emulsification method

[0043] A solution of 35 g agarose in 490 ml water was prepared at 95 ° C and subsequently cooled to 70 ° C after adding 7.0 mM phosphate to obtain pH 7.0. A solution of emulsifier in 850 ml toluene was prepared and heated to 60 ° C in a 3L thermostatic jacketed cylindrical glass reactor. The agarose solution was added to the reactor under stirring at 80 rpm with an overhead stirrer and continued to stir at 60 ° C with gradually increasing rpm until the agarose droplet size was about 100 μm, which was evaluated by the sample taken and analyzed by laser diffraction. Prior to analysis, these samples were quickly cooled with ice to avoid any coalescence / aggregation. The temperature of the reactor jacket was then reduced to 20 ° C to solidify the agarose droplets. The obtained agarose beads were washed with toluene and / or water, and the washings were decanted while recovering the agarose beads as precipitates.

[0044] Evaluation Method

[0045] Mastersizer 3000 laser diffractometer (Malvern Panalytical) is used to measure the particle size distribution of agarose beads with ethylcellulose as dispersant in ethanol dispersion. Distribution is plotted as a curve of differential volume to diameter, and the mode of each distribution is calculated by instrument. The mode is the peak of distribution, i.e. the highest peak seen in the distribution curve. Therefore, the mode represents the particle size most commonly found in the distribution. Before and after the emulsion is cooled, both samples are taken, and the difference between the mode after cooling and the mode before cooling is expressed as Δ mode. This is a measure of the increase in particle size during cooling, indicating that aggregation and / or aggregation occurs during the sensitive cooling stage.

[0046] The beads were also evaluated visually under a microscope for inclusions in the beads (which may result from oil-in-water-in-oil double emulsion formation) and sphericity, where deviations from sphericity may be due to partial coalescence of the droplets. Figure 2-4 Examples of aggregates, non-spherical beads, and beads with inclusions are shown.

[0047] Emulsifier

[0048] Table 1. Emulsifiers used

[0049] Product Name supplier Chemical structure Fatty alcohol part Ethylene oxide unit (n) Lubrhophos LF-800 Solvay C10-16 Ethoxylated / Propoxylated Phosphate Esters C10-16 Lubrhophos LB-400 Solvay Polyoxyethylene oleyl ether phosphate C16-18:1 5 Rhodafac PA / 32 Solvay Polyoxyethylene oleyl ether phosphate C16-18:1 2 Rhodafac PA / 35 Solvay Polyoxyethylene oleyl ether phosphate C16-18:1 5 Crodafos SG-LQ Croda PPG-5-Ceteth-10 Phosphate C16 10(PO5) Crodafos O10A Croda Oleth-10-phosphate C18:1 10 Crodafos O3A Croda Oleth-3 Phosphate C18:1 3 Crodafos CS2A Croda Ceteareth-2 Phosphate C16-18 2 Lakeland PAE 185 Lakeland Ethoxylated stearyl alcohol 5 EO phosphate C18 5 Hostaphat KL340D Clariant Lauryl polyethoxy (4EO) phosphate C12 4 Rhodafac RM-510 Solvay Dinonylphenol ethoxylate phosphate 10 SPAN 60 Croda Sorbitan monostearate SPAN 80 Croda Sorbitan monooleate SPAN 120 Croda Sorbitan Isostearate

[0050] Example 1 - Emulsifier Comparison

[0051] Table 2. Emulsification results

[0052] Emulsifier Emulsifier concentration in toluene phase (wt. / vol%) Contents(Y / N) Spherical(Y / N) Emulsification time (min) Maximum stirring speed (rpm) Mode before cooling (µm) Δ Mode(µm) Lubrhophos LF-800 0.016 N Y 1450 234 57 Lubrhophos LF-800 0.05 N Y 157 1350 103 35 Lubrhophos LF-800 0.08 N Y 105 1450 96 8 Lubrhophos LF-800 0.16 N Y 800 19 17 Lubrhophos LF-800 0.20 N Y 800 15 6 Lubrhophos LB-400 0.016 Y N 106 1500 177 143 Lubrhophos LB-400 0.05 N Y 168 1350 106 86 Lubrhophos LB-400 0.16 N Y 100 1450 97 17 Rhodafac PA / 32 0.16 Y Y 151 1600 108 20 Rhodafac PA / 35 0.16 N Y 108 1550 108 12 Crodafos SG-LQ 0.16 N N 144 1600 115 98 Crodafos SG-LQ 0.16 N N 118 1550 103 45 Crodafos O10A 0.16 Y N 148 1650 161 548 Crodafos O3A 0.16 N Y 87 1300 102 5 Crodafos CS2A 0.16 N Y 70 1250 21 2 Lakeland PAE 185 0.16 Y N 153 1650 125 130 Hostaphat KL340D 0.16 N Y 64 1200 112 34 Rhodafac RM-510 0.16 N Y 74 1350 96 25 Span 60 0.2 Y N 218 1550 778 176 Span 80 0.1 Y N 156 1250 559 Span 80 0.2 Y N 148 1200 810 Span 80 0.5 Y N 195 1600 218 123 Span 80 2 N Y 70 1250 99 5 Span 120 0.2 Y N 139 1250 907 Span 120 0.5 N Y 118 1450 413 -102 Span 120 2.3 N Y 111 1450 100 7

[0053] Basically, all the materials tested act as emulsifiers. However, there are differences between them. Non-ionic emulsifiers, such as the three sorbitan esters, must be used at a concentration of 2% to obtain good results. This means that a large amount of emulsifier must be washed out of the beads during the recovery process, which is undesirable, especially because those emulsifiers are insoluble in water and must be washed out with solvents. Phosphate esters can be used in lower concentrations (less than 0.2%) and are generally soluble in water. Particularly good results were obtained with Lubrhophos LF-800, Lubrhophos LB-400, Rhodafac PA / 35, Crodafos CS2A and CrodafosO3A, and Lubrhophos LF-800 and Crodafos O3A were selected as the best candidates. Rhodafac RM-510 also obtained good results, but was not selected for environmental reasons because it is based on alkylphenol ethoxylates.

[0054] Example 2-2-Methylcyclohexanone as solvent

[0055] Two experiments were performed as above, but 2-methylcyclohexanone was used instead of toluene. The emulsifier was Lubrhophos LF-800 and the amount of emulsifier was 0.017 wt. / vol. %. %, 0.23 wt. / vol. %, respectively. The target particle size of 100 µm was achieved and the visual appearance of the beads was good. However, some aggregation occurred during cooling.

[0056] This written description uses examples to disclose the invention, including the best mode, and also to enable those skilled in the art to practice the invention, including making and using any device or system and performing any combined methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. If such other examples have structural elements that are not different from the literal language of the claims, or if they include equivalent structural elements that are not substantially different from the literal language of the claims, it is intended that such other examples are within the scope of the claims. All patents and patent applications mentioned herein are incorporated herein by reference in their entirety, as if individually incorporated.

Claims

1. A method for making agar or agarose beads, comprising the following steps: a) providing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature of 40-100° C.; b) providing an oil phase comprising a water-immiscible solvent and an emulsifier at a temperature of 40-100° C.; c) emulsifying the aqueous phase in the oil phase to form a water-in-oil emulsion; d) cooling the water-in-oil emulsion to a temperature below the gelling temperature of the agar or agarose to form a dispersion of solidified agar or agarose beads; and e) recovering agar or agarose beads from the dispersion, The emulsifier comprises a phosphoric acid ester of an alkoxylated fatty alcohol.

2. The method of claim 1, wherein the water-immiscible solvent comprises toluene or xylene.

3. The method of claim 1 or 2, wherein the oil phase comprises less than 0.1% by weight of alkylphenol or alkylphenol derivatives.

4. The method of claim 1 or 2, wherein the oil phase does not contain alkylphenol and alkylphenol derivatives.

5. The method of claim 1 or 2, wherein the emulsifier comprises a mixture of phosphoric acid monoesters and phosphoric acid diesters of the alkoxylated fatty alcohol.

6. The method of claim 1 or 2, wherein the fatty alcohol comprises one or more C 10 -C 20 Linear or branched primary or secondary alkanols or enols.

7. The method of claim 1 or 2, wherein the alkoxylated fatty alcohol has the following structure R1-O-(R2-O) n -H(I) in: R1 is a saturated or unsaturated, linear or branched aliphatic C 10 -C 20 hydrocarbon, R2 is -CH2-CH2-, or a mixture of -CH2-CH2- and -CH2(CH3)-CH2-, and n is 2-20.

8. The method of claim 1 or 2, wherein the emulsifier comprises a mixture of the following substances: R1-O-(R2-O) n -P(O)(OH)-OH(II) and R1-O-(R2-O) n -P(O)(OH)-(O-R2) n -O-R1(III) in: R1 is a saturated or unsaturated, linear or branched aliphatic C 10 -C 20 hydrocarbon, R2 is -CH2-CH2-, or a mixture of -CH2-CH2- and -CH2(CH3)-CH2-, and n is 2-20.

9. The method of claim 8, wherein R1 is a saturated or unsaturated linear aliphatic C 10 -C 18 hydrocarbon.

10. The method of claim 8, wherein n is 2-10.

11. The method of claim 8, wherein n is 2-5.

12. The method of claim 1 or 2, wherein the emulsifier comprises C 10 -C 16 A mixture of phosphoric acid monoesters and phosphoric acid diesters of mixed ethylene oxide + propylene oxide adducts of alkanols.

13. The method of claim 1 or 2, wherein the emulsifier comprises oleth-3-phosphate.

14. The method of claim 1 or 2, wherein the emulsifier comprises Lubrhophos TM LF-800.

15. The method of claim 1 or 2, wherein the emulsifier comprises Crodafos TM O3A.

16. The method of claim 1 or 2, wherein the aqueous solution of agar or agarose comprises 1-8% by weight of agar or agarose.

17. The method of claim 1 or 2, wherein the oil phase comprises 0.01-2 wt% of the emulsifier.

18. The method of claim 1 or 2, wherein step c) comprises mixing the water phase and the oil phase in a stirred vessel to form a water-in-oil emulsion.

19. The method of claim 18, wherein step c) further comprises passing the water-in-oil emulsion through a rotor-stator mixer to reduce the droplet size of the water-in-oil emulsion.

20. The method of claim 18, wherein step c) further comprises passing the water-in-oil emulsion through a static mixer to reduce the droplet size of the water-in-oil emulsion.

21. The method of claim 18, wherein step c) further comprises passing the water-in-oil emulsion through a porous membrane to reduce the droplet size of the water-in-oil emulsion.

22. The method of claim 1 or 2, wherein step c) comprises passing the aqueous phase through a porous membrane into the oil phase to form a water-in-oil emulsion.

23. The process of claim 1 or 2, wherein step d) is performed in a stirred vessel.

24. The method of claim 1 or 2, wherein step d) is performed by passing the water-in-oil emulsion through a conduit having a longitudinally decreasing temperature gradient.

25. The method of claim 1 or 2, wherein step e) comprises adding water or an aqueous solution to the dispersion, decanting the oil phase, and recovering the agar or agarose beads as a precipitate.

26. The method of claim 1 or 2, wherein step e) comprises washing the agar or agarose beads with water or an aqueous solution to remove residual emulsifier.

27. The method of claim 1 or 2, further comprising the step f) of cross-linking the agar or agarose beads after step e).

28. The method of claim 1 or 2, further comprising the step g) of coupling a ligand to the agar or agarose beads after step e).

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