阅读说明：本技术 制造琼脂或琼脂糖珠的方法 (Method for producing agar or agarose beads ) 是由 S·林德贝格 J·古斯塔夫松 L·卡尔松 A·哈格瓦尔 E·林格贝格 D·扬松 于 2020-04-28 设计创作，主要内容包括：本发明公开了制造琼脂或琼脂糖珠的方法,包括以下步骤：a)在40-100℃的温度下提供包含琼脂或琼脂糖的水溶液的水相；b)在40-100℃的温度下提供包含水不混溶性溶剂和乳化剂的油相；c)将所述水相在所述油相中乳化以形成油包水乳液；d)将所述油包水乳液冷却至低于所述琼脂或琼脂糖的胶凝温度的温度,以形成固化的琼脂或琼脂糖珠的分散体；以及e)从所述分散体中回收琼脂或琼脂糖珠,其中所述乳化剂包含烷氧基化脂肪醇的磷酸酯。(The invention discloses a method for manufacturing agar or agarose beads, which comprises the following steps: a) providing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature of 40-100 ℃; b) providing an oil phase comprising a water-immiscible solvent and an emulsifier at a temperature of 40-100 ℃; 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 gelation 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, wherein the emulsifier comprises a phosphate ester of an alkoxylated fatty alcohol.)

1. A method of 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 ℃;

b) providing an oil phase comprising a water-immiscible solvent and an emulsifier at a temperature of 40-100 ℃;

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 gelation 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,

wherein the emulsifier comprises a phosphate 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 wt% of alkylphenols or alkylphenol derivatives.

4. The method of any of the preceding claims, wherein the oil phase is free or substantially free of alkylphenols and alkylphenol derivatives.

5. The method of any preceding claim, wherein the emulsifier comprises a mixture of mono-and di-phosphate esters of the alkoxylated fatty alcohol.

6. The method of any one of the preceding claims, wherein the fatty alcohol comprises one or more C₁₀-C₂₀Linear or branched primary or secondary alkanols or alkenols.

7. The method of any preceding claim, wherein the alkoxylated fatty alcohol has the structure

R₁ – O – (R₂-O)_n-H (I)

Wherein:

R₁is saturated or unsaturated, linear or branched aliphatic C₁₀-C₂₀A hydrocarbon(s) is (are) present,

R₂is-CH₂-CH₂-, or-CH₂-CH₂-and-CH₂ (CH₃) -CH₂A mixture of (A) and (B), and

n is 2 to 20.

8. The method of any preceding claim, wherein the emulsifier comprises a mixture of

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)

wherein:

R₁is saturated or unsaturated, linear or branched aliphatic C₁₀-C₂₀A hydrocarbon(s) is (are) present,

R₂is-CH₂-CH₂-, or-CH₂-CH₂-and-CH₂ (CH₃) -CH₂A mixture of (A) and (B), and

n is 2 to 20.

9. The method of claim 8, wherein R₁Is a saturated or unsaturated linear aliphatic C₁₀-C₁₈A hydrocarbon.

10. The method of claim 8 or 9, wherein n is 2-10, such as 2-5.

11. The method of any one of the preceding claims, wherein the emulsifier comprises C₁₀-C₁₆A mixture of phosphoric acid mono-and diesters of mixed ethylene oxide + propylene oxide adducts of alkanols.

12. The method of any preceding claim, wherein the emulsifier comprises oleyl polyether-3-phosphate (oleth-3-phosphate).

13. The method of any one of the preceding claims, wherein the emulsifier comprises lubrophos^TM LF-800。

14. The method of any of the preceding claims, wherein the emulsifier comprises Crodafos^TM O3A。

15. The method of any one of the preceding claims, wherein the aqueous solution of agar or agarose comprises 1-8% by weight of agar or agarose.

16. The method of any of the preceding claims, wherein the oil phase comprises 0.01-2 wt% of the emulsifier.

17. The method of any of the preceding claims, wherein step c) comprises mixing the aqueous phase and the oil phase in a stirred vessel to form a water-in-oil emulsion.

18. The method of claim 17 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.

19. The method of claim 17 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.

20. The method of claim 17 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.

21. The method of any one of claims 1-16, wherein step c) comprises passing the aqueous phase through a porous membrane into the oil phase to form a water-in-oil emulsion.

22. The process of any of the preceding claims, wherein step d) is carried out in a stirred vessel.

23. The process of any one of claims 1 to 21, wherein step d) is carried out by passing the water-in-oil emulsion through a conduit having a longitudinally decreasing temperature gradient.

24. The process of any one of the preceding claims, 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.

25. The method of any one of the preceding claims, wherein step e) comprises washing the agar or agarose beads with water or an aqueous solution to remove residual emulsifier.

26. The method of any one of the preceding claims, further comprising step f) cross-linking the agar or agarose beads after step e).

27. The method of any one of the preceding claims, further comprising step g) of coupling a ligand to the agar or agarose beads after step e).

28. Agar or agarose beads obtainable by the method of any one of the preceding claims.

Technical Field

The present invention relates to agar/agarose beads, and more particularly to methods of making agar or agarose beads. The invention also relates to an emulsifier suitable for use in the process.

Background

Agarose beads have been used as stationary phases in the chromatographic separation of proteins and other biological macromolecules for decades. They are typically prepared by reverse phase suspension gelation, in which 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 below the gelation temperature of agarose/agar to produce gel beads, which can then be recovered and used for separation purposes. These methods are described, for example, in S Hjerten: 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, which is also incorporated by reference in its entirety, where agarose beads are converted into agarose beads by hydrolysis of the sulfate groups after gelation.

In this process, it is necessary to use emulsifiers to stabilize the W/O emulsion. Emulsifiers are also important for the size distribution of the resulting beads and their shape. Furthermore, the emulsifier should be easy to remove from the beads by washing, and it should be environmentally friendly and not generate any toxic leachables when the beads are used in the manufacture of a medicament.

Previously disclosed emulsifiers lack several of these aspects and, therefore, additional emulsifiers are needed.

Disclosure of Invention

One aspect of the present invention is to provide a method for manufacturing agar or agarose beads. This is achieved by a method comprising the steps of:

a) providing an aqueous phase comprising an aqueous solution of agar or agarose at a temperature of 40-100 ℃;

b) providing an oil phase comprising a water-immiscible solvent and an emulsifier at a temperature of 40-100 ℃;

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

d) cooling the W/O emulsion to a temperature below the gelation 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,

wherein the emulsifier comprises a phosphate ester of an alkoxylated fatty alcohol.

One advantage is that aggregation of the beads in step d) is prevented, resulting in well-dispersed high sphericity beads. Other advantages are that the emulsifier is water soluble, easy to remove by water washing, and it is free of endocrine disruptors such as alkylphenol derivatives.

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

Further suitable embodiments of the invention are described in the dependent claims.

Drawings

FIG. 1 shows an example of well dispersed spherical agarose beads formed from a well stabilized W/O agarose emulsion.

Fig. 2 shows an example of agarose beads with aggregates (indicated by arrows) that may form during cooling of the W/O agarose emulsion.

Figure 3 shows an example of an agarose bead with non-spherical, partially coalesced beads (indicated by arrows).

Figure 4 shows an example of agarose beads with spherical inclusions (indicated by arrows) in the beads, which are caused by the formation of an O/W/O double emulsion.

Detailed Description

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

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

b) the oil phase comprising the water-immiscible solvent and the emulsifier is provided at a temperature of from 40 to 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. To facilitate efficient solvent recovery, the solvent may have a boiling point in the range of about 90-170 ℃ at atmospheric pressure, such as 90-150 ℃ or 100-120 ℃. 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-. Higher boiling solvents, such as mineral or vegetable oils, 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 comprise a mixture of several emulsifiers. The concentration of emulsifier (or total emulsifier concentration) in the oil phase may be, for example, 0.01 to 2 wt%, for example 0.015 to 1 wt%. Suitably, none of the emulsifiers comprise an alkylphenol or alkylphenol derivative. The oil phase may, for example, comprise less than 0.1 wt%, such as less than 0.01 or less than 0.001 wt% of alkylphenols or alkylphenol derivatives. Even without alkylphenols and alkylphenol derivatives;

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 a W/O emulsion. This step may further comprise 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 an emulsion, the aqueous phase may enter the oil phase through a porous membrane or sieve to form a W/O emulsion;

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

e) recovering the agar or agarose beads from the dispersion. 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 materials. Washing with organic solvents can also be used to remove emulsifier residues and other leachables.

After step e), the beads may be crosslinked in step f) by adding a crosslinking agent, for example 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 ligand to non-crosslinked beads.

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

Such emulsifiers include phosphate esters of alkoxylated fatty alcohols. Typically, it may comprise a mixture of phosphoric monoesters and phosphoric diesters of alkoxylated fatty alcohols. The fatty alcohol may compriseOne or more C₁₀-C₂₀The linear or branched, primary or secondary alkanol or enol and/or ethoxylated fatty alcohol may have structure I

R₁ – O – (R₂-O)_n-H (I)

Wherein:

R₁is saturated or unsaturated, linear or branched aliphatic C₁₀-C₂₀Hydrocarbons, e.g. saturated or unsaturated linear aliphatic C₁₀-C₁₈A hydrocarbon(s) is (are) present,

R₂is-CH₂-CH₂-, or-CH₂-CH₂-and-CH₂ (CH₃) -CH₂A mixture of (A) and (B), and

n is 2 to 20, such as 2 to 10 or 2 to 5.

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

R₁-O- (R₂-O) _n-P (O) (OH) -OH (II)

R₁-O- (R₂-O) _n-P (O) (OH) - (O-R₂) _n-O-R₁ (III)

wherein:

R₁is saturated or unsaturated, linear or branched aliphatic C₁₀-C₂₀Hydrocarbons, e.g. saturated or unsaturated linear aliphatic C₁₀-C₁₈A hydrocarbon(s) is (are) present,

R₂is-CH₂-CH₂-, or-CH₂-CH₂-and-CH₂ (CH₃) -CH₂A mixture of (A) and (B), and

n is 2 to 20, such as 2 to 10 or 2 to 5.

Both the phosphoric monoester II and the diester III are acidic compounds having dissociable acidic hydrogens. Thus, the phosphate ester emulsifiers of the present invention may be provided in acid form or in neutralized form (e.g., sodium or potassium or ammonium salts). If an emulsifier in acid form is used, a base may be suitably added to the aqueous phase to adjust the pH to near neutral, since agar and agarose are susceptible to degradation under acidic conditions. In addition to the monoesters II and III, the emulsifiers may also comprise the corresponding phosphoric acid triesters of alkoxylated fatty alcohols and free unesterified alkoxylated fatty alcohols. Both compounds are non-acidic and are generally present in small amounts, e.g. < 25% or less than 15% by weight of the emulsifier.

In particular, the emulsifier may comprise C₁₀-C₁₆A mixture of phosphoric acid mono-and diesters of mixed ethylene oxide + propylene oxide adducts of alkanols. Such products may be Lubrophos^TMLF-800 (Solvay) is available commercially. Alternatively, the emulsifier may comprise a mixture of a phosphoric acid monoester and a phosphoric acid diester of an ethoxylated oleyl alcohol having n =3 (average number of ethylene glycol units per oleyl alcohol). Such products are generally known under the INCI (International nomenclature for cosmetic ingredients) name oleyl polyether-3-phosphate (oleth-3-phosphate) and may be given the trade name Crodafos^TMO3A (Croda) is commercially available.

Examples

Emulsification process

A solution of 35g agarose in 490 ml water was prepared at 95 ℃ and subsequently cooled to 70 ℃ after addition of 7.0 mM phosphate, giving a pH of 7.0. A solution of the emulsifier in 850 ml of toluene was prepared and heated to 60 ℃ in a 3L thermostatically jacketed cylindrical glass reactor. The agarose solution was added to the reactor with stirring with an overhead stirrer at 80 rpm and stirring was continued at gradually increasing rpm at 60 ℃ until the agarose droplet size was about 100 μm, which was evaluated from the removed sample and analyzed by laser diffraction. Prior to analysis, the samples were rapidly cooled with ice to avoid any coalescence/aggregation. The temperature of the reactor jacket was then lowered to 20 ℃ to solidify the agarose droplets. The resulting agarose beads were washed with toluene and/or water, and the washing solution was decanted while the agarose beads were recovered as a precipitate.

Evaluation method

The particle size distribution of agarose beads in an ethanol dispersion with ethylcellulose as a dispersant was measured using a Mastersizer 3000 laser diffractometer (Malvern Panalytical). The distributions are plotted as differential volume versus diameter curves, and the mode of each distribution is calculated by the instrument. The mode is the peak of the distribution, i.e. the highest peak seen in the distribution curve. Thus, the mode represents the particle size most commonly found in a distribution. Samples were taken both before and after the emulsion was cooled and the difference between the mode after cooling and the mode before cooling was expressed as delta mode. This is a measure of the increase in particle size during cooling, indicating that coalescence and/or aggregation occurs during the sensitive cooling stage.

The beads were also evaluated visually in a microscope with respect to the inclusion in the bead (which may result from oil-in-water-in-oil double emulsion formation) and the spherical shape, where deviations from the spherical shape may be due to partial coalescence of the droplets. FIGS. 2-4 show examples of aggregates, non-spherical beads, and beads with inclusions.

Emulsifier

TABLE 1 emulsifiers used

Product name	Suppliers of goods	Chemical structure	Fatty alcohol moieties	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-Centeth-10 phosphate	C16	10(PO5)
Crodafos O10A	Croda	Oleyl polyether-10 phosphate (oleth-10-phosphate)	C18:1	10
					Crodafos O3A	Croda	Oleyl polyether-3 phosphate	C18:1	3
Crodafos CS2A	Croda	Ceteareth-2 phosphate	C16-18	2
					Lakeland PAE 185	Lakeland	Phosphoric acid esters of ethoxylated stearyl alcohol 5 EO	C18	5
Hostaphat KL340D	Clariant	Lauryl Polyethoxy (4EO) phosphate	C12	4
					Rhodafac RM-510	Solvay	Dinonylphenol ethoxylate phosphate esters		10
SPAN 60	Croda	Sorbitan monostearate
					SPAN 80	Croda	Sorbitan monooleate
SPAN 120	Croda	Sorbitan isostearate

Example 1 emulsifier comparison

TABLE 2 emulsification results

Emulsifier	Emulsifier concentration in toluene phase (wt./vol%)	Inclusions (Y/N)	Spherical (Y/N)	Emulsification time (min)	Maximum stirring speedDegree (rpm)	Mode before cooling (mu m)	Delta mode (mum)
								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

Essentially, all materials tested acted as emulsifiers. However, there are differences between them. Nonionic emulsifiers, such as the three sorbitan esters, must be used at a concentration of 2% to give good results. This means that large amounts of emulsifiers have to be washed out of the beads during the recovery process, which is undesirable, in particular because those emulsifiers are insoluble in water and have to be washed out with solvents. Phosphate esters can be used at lower concentrations (below 0.2%) and are generally water soluble. Particularly good results were obtained with Lubrophos LF-800, Lubrophos LB-400, Rhodafac PA/35, Crodafos CS2A and Crodafos O3A, with Lubrophos LF-800 and Crodafos O3A being selected as the best candidates. Rhodafac RM-510 also gave good results, but was not chosen for environmental reasons because it is based on alkylphenol ethoxylates.

Example 2-2-Methylcyclohexanone as solvent

Two experiments were performed as above, but with 2-methylcyclohexanone instead of toluene. The emulsifier is Lubrophos LF-800, and the amount of the emulsifier is 0.017 wt./vol. % and 0.23 wt./vol.%. A target particle size of 100 μm is achieved and the visual appearance of the beads is good. But some agglomeration occurs during cooling.

This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated 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. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. All patents and patent applications mentioned herein are incorporated by reference in their entirety as if individually incorporated.