Patent Publication Number: US-2022219141-A1

Title: Method of Manufacturing Agar or Agarose Beads

Description:
TECHNICAL FIELD OF THE INVENTION 
     The present invention relates to agar/agarose beads, and more particularly to a method for manufacturing agar or agarose beads. The invention also relates to emulsifiers suitable for use in the method. 
     BACKGROUND OF THE INVENTION 
     Agarose beads have for several decades been used as a stationary phase in chromatographic separations of proteins and other biomacromolecules. They are typically prepared by inverse suspension gelation, where 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 the agarose/agar to create gel beads, which can then be recovered and used for separation purposes. Such processes are described e.g. in S Hjertén: Biochim Biophys Acta 79(2), 393-398 (1964), WO1989011493A1 and US20180171484, hereby incorporated by reference in their entireties. A variant where agar beads are converted to agarose beads by hydrolysis of sulfate groups post-gelation is described in US20100084345, also incorporated by reference in its entirety. 
     In the process it is essential to use an emulsifier for stabilization of the W/O emulsion. The emulsifier will also be important for the size distribution of the resulting beads and for their shape. Further, the emulsifier should be easy to remove from the beads by washing and it should be environmentally friendly and not give rise to any toxic leachables when the beads are used for manufacturing of pharmaceuticals. 
     The emulsifiers previously disclosed are lacking in several of these aspects and accordingly, there is a need for further emulsifiers. 
     SUMMARY OF THE INVENTION 
     One aspect of the invention is to provide a method for the manufacturing agar or agarose beads. This is achieved with a method comprising the steps of: 
     a) providing a water 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 water phase in the oil phase to form a water-in-oil (W/O) emulsion; 
     d) cooling the W/O emulsion to a temperature below a 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 during step d) is prevented, such that well-dispersed beads of high sphericity are produced. Further advantages are that the emulsifier is water-soluble, facilitating removal by water washing, and that it is free from endocrine disruptors such as alkyl phenol derivatives. 
     A further aspect of the 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 a W/O agarose emulsion. 
         FIG. 3  shows an example of agarose beads with non-spherical, partially coalesced beads (indicated by arrows). 
         FIG. 4  shows an example of agarose beads with spherical inclusions in the beads (indicated by arrows), caused by O/W/O double emulsion formation. 
     
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
     In one aspect, the present invention discloses a method for the manufacturing agar or agarose beads. The method comprises the steps of: 
     a) Providing a water phase comprising an aqueous solution of agar or agarose at a temperature of 40-100° C. The water phase may e.g. comprise 1-8 wt. % agar or agarose, such as 2-7 wt. % agar or agarose, e.g. about 2 wt. %, about 4 wt. % or about 6 wt. %. The water phase can further comprise one or more buffer components such as a weak base. The agar or agarose may be native agar, native agarose or a derivative of agar or agarose, such as e.g. allyl agarose or hydroxyethyl agarose, further described in U.S. Pat. No. 6,602,990 and U.S. Pat. No. 7,396,467, hereby incorporated by reference in their entireties; 
     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 can e.g. be a hydrocarbon, an ester or a ketone. To facilitate an efficient solvent recovery, the solvent may have a boiling point in the range of about 90-170° C. , such as 90-150° C., or 100-120° C., at atmospheric pressure. It can e.g. be toluene (b.p. 111° C.) or xylene (b.p. about 140° C.: 139° C. for m-xylene and 144° C. for o-xylene). Alternatively, it can be a cyclic ketone, such as 2-methylcyclohexanone (b.p. 162-163° C.). It is also possible to use higher-boiling solvents, such as mineral oils or vegetabilic oils, although the solvent may then be more difficult to recover. The nature of the emulsifier is further described below. The emulsifier may be a single emulsifier or the oil phase may comprise a mixture of several emulsifiers. The concentration of the emulsifier (or the total emulsifier concentration) in the oil phase may e.g. be 0.01-2 wt. %, such as 0.015-1 wt. %. Suitably, none of the emulsifiers comprises alkyl phenols or alkyl phenol derivatives. The oil phase can e.g. comprise less than 0.1 wt %, such as less than 0.01 or less than 0.001 wt % alkyl phenols or alkyl phenol derivatives. It can even be devoid of alkyl phenols and alkyl phenol derivatives; 
     c) Emulsifying the water phase in the oil phase to form a water-in-oil (W/O) emulsion. The emulsification may comprise mixing the water phase and the oil phase in an agitated vessel to form a W/O emulsion. The step may further comprise passing the W/O emulsion through a rotor-stator mixer, a static mixer or a porous membrane to reduce a droplet size of the W/O emulsion. In an alternative way of forming the emulsion, the water phase can be passed through a porous membrane or sieve plate into the oil phase to form a W/O emulsion; 
     d) Cooling the W/O emulsion to a temperature below a gelation temperature of the agar or agarose to form a dispersion of solidified agar or agarose beads. This step can be performed by gradually cooling the emulsion in an agitated vessel or it can be performed in continuous mode by passing the W/O emulsion through a conduit with a longitudinally decreasing temperature gradient; 
     e) Recovering 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 sediment. The beads may further be washed with water or an aqueous solution to remove residual emulsifier and/or other substances. Washing with organic solvents can also be applied for removal of emulsifier residues and other leachables. 
     After step e), the beads may be crosslinked in a step f), by adding a crosslinking agent, e.g. epichlorohydrin. They may further be functionalized with ligands in a step g), where the ligands are covalently coupled, using methods known to the skilled person. Step g) can suitably be performed after step f), although it is also possible to couple ligands on non-crosslinked beads. 
     The beads prepared by the process can be used in chromatographic separation processes or in batch adsorption processes. They may e.g. have diameters (expressed as the volume-weighted median diameter d50,v) in the range of 5-500 μm, such as 10-350 μm or 30-120 p.m. 
     The emulsifier as mentioned above comprises a phosphate ester of an alkoxylated fatty alcohol. Typically, it can comprise a mixture of phosphate monoester and phosphate diester of the alkoxylated fatty alcohol. The fatty alcohol may comprise one or more C 10 -C 20  linear or branched, primary or secondary, alkanols or alkenols and/or the ethoxylated fatty alcohol may have a structure I 
         R   1 —O—(R 2 -O) n—H    (I)
 
     where: 
     R 1  is a saturated or unsaturated, linear or branched, aliphatic C 10 -C 20  hydrocarbon, such as a saturated or unsaturated linear aliphatic C 10 -C 18  hydrocarbon, 
     R 2  is —CH 2 -CH 2 — or a mixture of —CH 2 -CH 2 — and —CH 2 (CH 3 )—CH 2 —, and n is 2-20, such as 2-10 or 2-5. 
     This structure describes an ethoxylated or mixed ethoxylated/propoxylated fatty alcohol, with average alkoxylation degree of n. The alkoxylated fatty alcohol has then been converted into phosphate ester of structure II and III, where II is a phosphate monoester and III is a phosphate diester: 
       R 1 —O—(R 2 −O) n −P( O )−OH   (II)
 
       R 1 —O—(R 2 −O) n −P(O)−(O−R 2 ) n —O−R 1    (III)
 
     where: 
     R 1  is a saturated or unsaturated, linear or branched, aliphatic C 10 -C 20  hydrocarbon, such as a saturated or unsaturated linear aliphatic C 10 -C 18  hydrocarbon, R 2  is —CH 2 -CH 2 — or a mixture of —CH 2 -CH 2 — and —CH 2 (CH 3 )-CH 2 —, and n is 2-20, such as 2-10 or 2-5. 
     Both the phosphate monoester II and the diester III are acidic compounds, with acidic hydrogens that can be dissociated. The phosphate ester emulsifiers of the invention can thus be supplied either in acid form or in neutralized form (e.g. as sodium salts or alternatively as potassium or ammonium salts). If emulsifiers in acid form are used, a base may suitably be added to the water phase to adjust the pH to near neutral, as agar and agarose are sensitive to degradation under acidic conditions. In addition to the monoester II and diester III, the emulsifier may also comprise the corresponding phosphate triester of the alkoxylated fatty alcohol and the free, non-esterified alkoxylated fatty alcohol. Both of these compounds are non-acidic and are usually present in minor amounts, such as &lt;25 wt% or less than 15 wt% of the emulsifier. 
     In particular, the emulsifier may comprise a mixture of phosphate monoester and phosphate diester of a mixed ethylene oxide+propylene oxide adduct of a C 10 -C 16  alkanol. Such a product is commercially available under the name of Lubrhophos™ LF-800 (Solvay). Alternatively, the emulsifier may comprise a mixture of phosphate monoester and phosphate diester of ethoxylated oleyl alcohol with n=3 (average number of ethylene glycol units per oleyl alcohol). Such a product is generally known under the INCI (International Nomenclature of Cosmetic Ingredients) name of oleth-3 phosphate and is commercially available under the trade name of Crodafos™ O3A (Croda). 
     EXAMPLES 
     Emulsification method 
     A solution of 35 g agarose in 490 ml of water was prepared at 95 ° C. and subsequently cooled to 70 ° C. after addition of 7.0 mM phosphate to give pH 7.0. A solution of emulsifier in 850 ml toluene was prepared and heated to 60 ° C. in a 3 L thermostated jacketed cylindrical glass reactor. Under agitation with an overhead agitator, the agarose solution was added to the reactor under 80 rpm agitation and the agitation was continued with stepwise increasing rpm at 60 ° C. until the agarose droplet size was approximately 100 μm, as assessed from samples removed and analysed by laser diffraction. These samples were rapidly cooled with ice to avoid any coalescence/aggregation before the analysis. The temperature of the reactor jacket was then lowered to 20 ° C. to solidify the agarose droplets. The resulting agarose beads were washed with toluene and/or water, the washing liquids were decanted while the agarose beads were recovered as a sediment. 
     Evaluation methods 
     The particle size distribution was measured using a Mastersizer 3000 laser diffraction instrument (Malvern Panalytical) for agarose beads in an ethanol dispersion with ethyl cellulose as a dispersant. The distributions were plotted as differential volume vs diameter curves and the mode of each distribution was calculated by the instrument. The mode is the peak of the distribution, i.e. the highest peak seen in the distribution curve. The mode thus represents the particle size most commonly found in the distribution. Samples were taken both before and after the cooling of the emulsions and the difference between the mode after cooling and the mode before cooling was denoted A mode. This is a measure of the particle size increase during cooling, indicative of coalescence and/or aggregation occurring during the sensitive cooling phase. 
     The beads were also evaluated visually in a microscope with respect to inclusions in the beads (which may result from oil-in-water-in-oil double emulsion formation) and spherical shape, where deviations from spherical shape may be due to partial coalescence of droplets. Examples of aggregates, non-spherical beads and beads with inclusions are shown in  FIGS. 2-4 . 
     Emulsifiers 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Emulsifiers used. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Fatty 
                 Ethylene 
               
               
                 Product 
                   
                   
                 alcohol 
                 oxide 
               
               
                 name 
                 Supplier 
                 Chemical structure 
                 moiety 
                 units (n) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Lubrhophos 
                 Solvay 
                 C10-16 ethoxylated/ 
                 C10-16 
                   
               
               
                 LF-800 
                   
                 propoxylated 
               
               
                   
                   
                 phosphate 
               
               
                 Lubrhophos 
                 Solvay 
                 Polyoxyethylene oleyl 
                 C16-18:1 
                 5 
               
               
                 LB-400 
                   
                 ether phosphate 
               
               
                 Rhodafac 
                 Solvay 
                 Polyoxyethylene oleyl 
                 C16-18:1 
                 2 
               
               
                 PA/32 
                   
                 ether phosphate 
               
               
                 Rhodafac 
                 Solvay 
                 Polyoxyethylene oleyl 
                 C16-18:1 
                 5 
               
               
                 PA/35 
                   
                 ether phosphate 
               
               
                 Crodafos 
                 Croda 
                 PPG-5-ceteth-10 
                 C16 
                 10(PO5) 
               
               
                 SG-LQ 
                   
                 phosphate 
               
               
                 Crodafos 
                 Croda 
                 Oleth-10 phosphate 
                 C18:1 
                 10 
               
               
                 O10A 
               
               
                 Crodafos 
                 Croda 
                 Oleth-3 phosphate 
                 C18:1 
                 3 
               
               
                 O3A 
               
               
                 Crodafos 
                 Croda 
                 Ceteareth-2 phosphate 
                 C16-18 
                 2 
               
               
                 CS2A 
               
               
                 Lakeland 
                 Lakeland 
                 Phosphate ester of 
                 C18 
                 5 
               
               
                 PAE 185 
                   
                 ethoxylated 
               
               
                   
                   
                 octadecanol 5 EO 
               
               
                 Hostaphat 
                 Clariant 
                 Lauryl polyethoxy 
                 C12 
                 4 
               
               
                 KL340D 
                   
                 (4EO) phosphate 
               
               
                 Rhodafac 
                 Solvay 
                 Phosphate ester of 
                   
                 10 
               
               
                 RM-510 
                   
                 dinonylphenol 
               
               
                   
                   
                 ethoxylate 
               
               
                 SPAN 60 
                 Croda 
                 Sorbitan monostearate 
               
               
                 SPAN 80 
                 Croda 
                 Sorbitan monooleate 
               
               
                 SPAN 120 
                 Croda 
                 Sorbitan isostearate 
               
               
                   
               
            
           
         
       
     
     Example 1—Emulsifier comparisons 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Emulsification results 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Conc. 
                   
                   
                   
                   
                   
                   
               
               
                   
                 emulsifier 
                   
                   
                   
                 Max 
               
               
                   
                 in toluene 
                   
                   
                   
                 stirring 
                 Mode prior 
                 Δ 
               
               
                   
                 phase 
                 Inclusions 
                 Spherical 
                 Emulsification 
                 speed 
                 to cooling 
                 mode 
               
               
                 Emulsifier 
                 (wt./vol %) 
                 (Y/N) 
                 (Y/N) 
                 time (min) 
                 (rpm) 
                 (μm) 
                 (μm) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Lubrhophos LF- 
                 0.016 
                 N 
                 Y 
                   
                 1450 
                 234 
                 57 
               
               
                 800 
               
               
                 Lubrhophos LF- 
                 0.05 
                 N 
                 Y 
                 157 
                 1350 
                 103 
                 35 
               
               
                 800 
               
               
                 Lubrhophos LF- 
                 0.08 
                 N 
                 Y 
                 105 
                 1450 
                 96 
                 8 
               
               
                 800 
               
               
                 Lubrhophos LF- 
                 0.16 
                 N 
                 Y 
                   
                 800 
                 19 
                 17 
               
               
                 800 
               
               
                 Lubrhophos LF- 
                 0.20 
                 N 
                 Y 
                   
                 800 
                 15 
                 6 
               
               
                 800 
               
               
                 Lubrhophos LB- 
                 0.016 
                 Y 
                 N 
                 106 
                 1500 
                 177 
                 143 
               
               
                 400 
               
               
                 Lubrhophos LB- 
                 0.05 
                 N 
                 Y 
                 168 
                 1350 
                 106 
                 86 
               
               
                 400 
               
               
                 Lubrhophos LB- 
                 0.16 
                 N 
                 Y 
                 100 
                 1450 
                 97 
                 17 
               
               
                 400 
               
               
                 Rhodafac PA/32 
                 0.16 
                 Y 
                 Y 
                 151 
                 1600 
                 108 
                 20 
               
               
                 Rhodafac PA/35 
                 0.16 
                 N 
                 Y 
                 108 
                 1550 
                 108 
                 12 
               
               
                 Crodafos SG- 
                 0.16 
                 N 
                 N 
                 144 
                 1600 
                 115 
                 98 
               
               
                 LQ 
               
               
                 Crodafos SG- 
                 0.16 
                 N 
                 N 
                 118 
                 1550 
                 103 
                 45 
               
               
                 LQ 
               
               
                 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 
                 0.16 
                 Y 
                 N 
                 153 
                 1650 
                 125 
                 130 
               
               
                 185 
               
               
                 Hostaphat 
                 0.16 
                 N 
                 Y 
                 64 
                 1200 
                 112 
                 34 
               
               
                 KL340D 
               
               
                 Rhodafac RM- 
                 0.16 
                 N 
                 Y 
                 74 
                 1350 
                 96 
                 25 
               
               
                 510 
               
               
                 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 
               
               
                   
               
            
           
         
       
     
     Basically, all the tested materials function as emulsifiers. There are however differences between them. The nonionic emulsifiers, as exemplified by the three sorbitan esters, have to be used at concentrations of 2% to give good results. This means that a high amount of emulsifier has to be washed out from the beads during the recovery, which is undesirable, in particular since those emulsifiers are not water soluble 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 Lubrhophos LF-800, Lubrhophos LB-400, Rhodafac PA/35, Crodafos CS2A and Crodafos O3A, with Lubrhophos LF-800 and Crodafos 03A selected as top candidates. Rhodafac RM-510 also gave good results but was deselected for environmental reasons, as it is based on alkylphenol ethoxylates. 
     Example 2—2-methylcyclohexanone as solvent 
     Two experiments were carried out as above, but with 2-methylcyclohexanone instead of toluene. 
     The emulsifier was Lubrhophos LF-800 and the emulsifier amounts were 0.017 wt./vol. % and 0.23 wt./vol. % respectively. The target particle size of 100 μm was reached and the visual appearance of the beads was good. Some aggregation occurred during cooling though. 
     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 in the text are hereby incorporated by reference in their entireties as if individually incorporated.