Patent Description:
Crosslinked polysaccharide beads are commonly used as stationary phases for chromatographic separation of proteins and other biomolecules. Such beads were introduced in the early <NUM>-ies (see e.g. <CIT>,) mainly for laboratory separation purposes. Since then their use has grown dramatically and crosslinked polysaccharide beads are now used routinely in large scale manufacturing processes for separation of many biopharmaceuticals such as monoclonal antibodies, plasma components, insulin and various recombinant proteins.

The most common way to prepare polysaccharide beads is by inverse suspension processes, where an aqueous solution of a polysaccharide is emulsified as a water-in-oil (w/o) emulsion in a continuous oil phase and the emulsion droplets are solidified either by crosslinking or by thermal gelation. Such processes are described in e.g. <CIT>, <CIT> and <CIT> which use chlorinated hydrocarbons or aromatic hydrocarbons as the oil phase. An issue here is that large scale use of halogenated hydrocarbons and aromatic hydrocarbons is currently being phased out for environmental reasons.

Accordingly there is a need for methods to manufacture polysaccharide beads without the use of halogenated or aromatic solvents.

One aspect of the invention is to provide an environmentally acceptable method of manufacturing polysaccharide beads. This is achieved with a method as defined in the claims. One advantage is that the method does not use halogenated or aromatic solvents. Further advantages are that spherical beads with good pore structures and mechanical properties can be obtained.

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

In one aspect, illustrated by <FIG>, the present invention discloses a method of manufacturing polysaccharide beads, comprising the steps of:.

The at least one organic solvent is an alicyclic ketone defined by Formula II. Alternatively, or additionally, the at least one organic solvent does not contain halogens (i.e. the molecules of the solvent do not contain halogen atoms) and has Hansen solubility parameter values in the ranges of δD = <NUM> - <NUM> MPa<NUM>/<NUM>, δP = <NUM> - <NUM> MPa<NUM>/<NUM> and δH = <NUM> - <NUM> MPa<NUM>/<NUM>. The oil phase can also comprise a mixture of halogen-free water-immiscible organic solvents, where the mixture has Hansen solubility parameter values in the ranges of 6D = <NUM> - <NUM> MPa<NUM>/<NUM>, δP = <NUM> - <NUM> MPa<NUM>/<NUM> and δH = <NUM> - <NUM> MPa<NUM>/<NUM>. Suitably, the content of halogenated solvents in the oil phase can be less than <NUM> mol %, such as less than <NUM>% or less than <NUM> %. The Hansen solubility parameters are discussed in detail in C M Hansen: The three dimensional solubility parameter and solvent diffusion coefficient - Their importance in surface coating formulation, Copenhagen <NUM>. δD is the dispersion force contribution to the solubility parameter (cohesive energy density) of a solvent, while δP is the polar force contribution and δH is the hydrogen bonding force contribution. Tables of Hansen solubility parameters for different solvents can be found e.g. in <NPL>.

In certain embodiments, illustrated by <FIG>, step iv) comprises crosslinking the polysaccharide. This can be accomplished e.g. by adding a crosslinking agent to the w/o emulsion. The crosslinking agent may e.g. be a compound with two electrophilic functionalities, which can react e.g. with two hydroxyl groups on the polysaccharide and cause crosslinking by the formation of covalently bonded links between polysaccharide chains. The hydroxyl groups are particularly nucleophilic at high pH conditions and it can be advantageous to use a high pH water phase in the method, e.g. by adding NaOH or other suitable alkali (e.g. KOH) to the water phase. The alkali (NaOH or KOH) concentration in the water phase may e.g. be at least <NUM>, such as <NUM>-<NUM> or <NUM>-<NUM>. Examples of electrophilic crosslinkers include epichlorohydrin, diepoxides and multifunctional epoxides, as well as divinylsulfone and halohydrins like <NUM>,<NUM>-dibromo-propanol-<NUM>. The crosslinker can suitably be added to the w/o emulsion, such that it dissolves in the oil phase and diffuses into the water phase droplets.

In some embodiments, step iv) comprises thermal gelation of the polysaccharide. In this case, the polysaccharide can be a hot-water soluble polysaccharide that forms a gel upon cooling. Examples of such polysaccharides are e.g. agar and agarose, which are soluble at temperatures of about <NUM> and higher but form solid gels upon cooling to e.g. about <NUM> or lower. In this case, steps i)-iii) can be performed at a temperature where the polysaccharide is soluble and in step iv) the temperature is lowered to a temperature below the gel point of the particular polysaccharide used.

In certain embodiments, at least one emulsifier is a cellulose derivative, such as a cellulose ester or a cellulose ether. Among cellulose esters, cellulose mixed esters, such as cellulose acetate butyrate can be particularly useful. Cellulose acetate butyrate (CAB) of different grades is commercially available, e.g. from Eastman Chemical Company (USA). The molecular weight of the CAB can suitably be <NUM>-<NUM> kDa, such as <NUM>-<NUM> kDa or <NUM>-<NUM> kDa, determined by gel permeation chromatography as the polystyrene equivalent number average molecular weight (Mn). The acetyl and butyryl contents can e.g. be <NUM>-<NUM> wt. % acetyl content and <NUM>-<NUM> wt. % butyryl content, such as a) <NUM>-<NUM> wt. % acetyl content and <NUM>-<NUM> wt. % butyryl content or b) <NUM>-<NUM> wt. % acetyl content and <NUM>-<NUM> wt. % butyryl content or c) <NUM>-<NUM> wt. % acetyl content and <NUM>-<NUM> wt. % butyryl content. An example of a cellulose ether useful as an emulsifier is ethyl cellulose.

In some embodiments at least one organic solvent is a C<NUM>-C<NUM> alicyclic ketone defined by Formula II,
<CHM>
<CHM>
<CHM>
wherein:.

In some embodiments, at least one organic solvent is defined by Formula II, with R<NUM> and R<NUM> defined as above. R<NUM> may e.g. be a C<NUM> alkylene group and R<NUM> may be a methyl group.

In certain embodiments, the organic solvent can be selected from the group consisting of <NUM>-methylcyclohexanone, <NUM>-methylcyclohexanone, <NUM>-methylcyclohexanone, cyclohexanone, diisobutyl ketone, methyl n-amyl ketone, methyl isoamyl ketone, methyl isobutyl ketone, cyclopentyl methyl ether and their mixtures. At least one organic solvent can e.g. be selected from the group consisting of <NUM>-methylcyclohexanone, <NUM>-methylcyclohexanone, <NUM>-methylcyclohexanone, cyclohexanone, methyl n-amyl ketone, methyl isoamyl ketone, methyl isobutyl ketone and cyclopentyl methyl ether, such as from the group consisting of <NUM>-methylcyclohexanone, <NUM>-methylcyclohexanone, <NUM>-methylcyclohexanone and cyclohexanone. In specific cases at least one organic solvent can be <NUM>-methylcyclohexanone.

A polysaccharide bead, or a plurality of polysaccharide beads, can be prepared by the method of any embodiment disclosed above. Disclosed herein is also a crosslinked polysaccharide bead, or a plurality of crosslinked polysaccharide beads, comprising at least <NUM> ppm, such as <NUM>-<NUM> ppm of a C<NUM>-C<NUM> aliphatic or alicyclic ketone or ether. This may e.g. constitute solvent residues from a manufacturing process as discussed above. The amount may be measured e.g. by headspace GC or GC analysis of extracts, using e.g. mass spectroscopy or flame ionization as detection method. The C<NUM>-C<NUM> aliphatic or alicyclic ketone or ether can be as defined by Formulas I, II or III as discussed above and it can e.g. be selected from the group consisting of <NUM>-methylcyclohexanone, <NUM>-methylcyclohexanone, <NUM>-methylcyclohexanone, cyclohexanone, diisobutylketone, methyl n-amylketone, methyl isoamyl ketone, methyl isobutyl ketone, cyclopentyl methyl ether and their mixtures.

In some embodiments, the bead or plurality of beads have a diameter of <NUM>-<NUM> in dry form. In the case of a plurality of beads, the diameter can be determined as the volume-weighted median diameter, d50v, e.g. by laser light diffraction or by electrozone sensing counting techniques.

In certain embodiments, the bead or plurality of beads comprise at least <NUM> mmol/g, or <NUM>-<NUM> mmol/g, covalently bonded charged groups, such as carboxymethyl-, sulfopropyl-, diethyl aminoethyl-, and/or diethyl-(<NUM>-hydroxy-propyl)aminoethyl- groups. Such groups are useful for ion exchange separations and groups like diethyl aminoethyl groups can also promote the growth of adherent cells when the beads are used as microcarriers in cell cultivation. The derivatisation with charged groups can be accomplished by methods well known in the art.

One or more polysaccharide beads as described above can be used for separation of a target biomolecule, such as a target protein, from impurities or contaminants.

One or more polysaccharide beads as described above can be used as microcarriers for cultivation of cells.

<NUM> dextran of Mw <NUM>-<NUM> kD was dissolved in <NUM> distilled water and simultaneously <NUM> <NUM>% sodium hydroxide (NaOH) was added under stirring. <NUM> sodium borohydride (NaBH<NUM>) was added to the solution.

<NUM> cellulose acetate butyrate (CAB) was added into a <NUM> glass reactor with an overhead agitator. The agitator was started and <NUM> <NUM>,<NUM>-dichloroethane (EDC) was added (the CAB concentration was thus <NUM>/ml EDC). The solution was stirred until the CAB had dissolved. The water phase was then added to the oil phase under agitation at <NUM> and the agitation was continued until a suitable droplet size had been reached, as judged from microscopy of small samples withdrawn. When a suitable size was obtained, the emulsion was immediately stabilised by adding <NUM> epichlorohydrin (ECH) to crosslink the dextran and thus solidifying the droplets. <NUM> minutes after the addition of ECH, <NUM> of EDC was added to make the reaction mixture less viscous and easier to agitate.

The crosslinking reaction was terminated after <NUM>±<NUM> by adding <NUM> acetone. The reaction mixture was then transferred to a <NUM> glass reactor containing <NUM> acetone. The solution was stirred for <NUM> and then the gel was subsequently allowed to sediment. The supernatant was decanted and the washing procedure was repeated <NUM> times with acetone and then <NUM> times with <NUM>% aqueous ethanol and <NUM> times with <NUM>% ethanol. The gel was then dried under vacuum at <NUM> for <NUM> and the resulting powder was sieved between <NUM> and <NUM> sieves.

The results from five repetitions of this experiment are shown in Table <NUM>, as samples <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

Approximately <NUM>-<NUM> grams of powder was weighed in a plastic bottle to which roughly <NUM> of either a <NUM>/ml NaCl solution or the running buffer (<NUM> NaCl+ <NUM> phosphate buffer, pH <NUM>) was added. The gel was left to swell and sediment for at least <NUM> hours. It was then washed several times and diluted in the running buffer to generate a <NUM>-<NUM>% gel slurry. Columns of <NUM> diameter and <NUM> height (GE Healthcare HR10/<NUM>) were packed with the initial flow rate of either <NUM> or <NUM>/min, and the final flow rate was either <NUM> or <NUM>/min. The packed columns were then tested in an effectiveness test and evaluated through selectivity tests with dextran standards and proteins as described in <NPL>. The test proteins were alpha chymotrypsinogen type II, bovine (Mw <NUM> kDa), ribonuclease A, bovine (Mw <NUM> kDa) and lysozyme, chicken (Mw <NUM> kDa), with <NUM> NaCl, <NUM> Sodium Phosphate, pH <NUM> as running buffer. KD data (i.e. the fraction of the bead volume accessible for a probe molecule of a particular size) for these proteins on the prototypes are shown in Table <NUM>.

Water regain (Wr) is the amount of water taken up inside the beads for <NUM> dry beads. A high Wr value indicates that the swollen gel is less dense and can separate high Mw target molecules. <NUM> of dry beads were equilibrated with <NUM> water for <NUM>. A <NUM> x <NUM> weighed centrifuge tube with a <NUM> bottom filter was filled with the gel slurry and centrifuged at <NUM> rpm for <NUM>. After determining the wet weight of the tube, it was dried over night at <NUM> and the dry weight was determined. The water regain was calculated as the drying weight loss (as ml water) per g dry gel.

The particle size distribution was measured in a Coulter Multisizer (Beckman Coulter), using the electrozone sensing technique.

After crosslinking, the beads were examined in a light microscope with phase contrast optics and the presence of surface dimples, inclusions, aggregates etc. was noted.

Emulsifications were carried out according to the reference example above, with other solvents replacing EDC, and in some cases with different types and/or concentrations of CAB. The CAB types used are specified in Table <NUM>. The beads were characterized as described above and the results are collated in Tables <NUM> and <NUM> and in the discussion below.

The solvents used were also analysed by NMR spectroscopy before and after emulsification model experiments performed in the absence of dextran and the emulsifier. The spectra before and after were essentially identical, showing that no degradation occurred during the reaction conditions used. This was in contrast to an ester solvent, t-butyl acetate, which although being a sterically hindered ester, was completely hydrolysed under the strongly alkaline conditions used.

The solvents evaluated produce beads that can be used for chromatography, as evidenced e.g. by their performance in the SEC analysis. The measured properties are in the same range as for the reference prototypes, which shows that the new solvents perform well. Due to the different interactions between the solvents and the CAB emulsifier, the CAB type and concentration had to be varied in order to get suitable oil phase viscosities. The beads were generally spherical (Figs <NUM>-<NUM>), but with the solvents <NUM>-MCH and <NUM>-MCH (particularly when used alone), some inclusions and dimples occurred on the beads.

Claim 1:
A method of manufacturing polysaccharide beads, comprising the steps of:
i) providing a water phase comprising an aqueous solution of a polysaccharide;
ii) providing an oil phase comprising at least one water-immiscible organic solvent and at least one oil-soluble emulsifier;
iii) emulsifying said water phase in said oil phase to form a water-in-oil (w/o) emulsion; and
iv) inducing solidification of said water phase in said w/o emulsion,
wherein said at least one organic solvent is an alicyclic ketone defined by Formula II,
<CHM>
wherein:
R<NUM> is a C<NUM>-C<NUM> alkylene group; and
R<NUM> is hydrogen or a C<NUM>-C<NUM> alkyl group.