Formulations for the production of porous polymer particles

Abstract

The present disclosure provides a formulation for the production of porous polymer particles. The formulation comprises a polymer; a salt; and at least one buffer. The salt is an antichaotropic salt, and the at least one buffer is present in a weight ratio of the at least one buffer to the polymer of at least 1:40.

Description

[Technical field]

[0001] The present disclosure relates to a formulation that can be used to produce porous polymer particles. In particular, the present disclosure relates to a formulation that can be used to produce porous polymer beads using a rotating disk in a continuous or semi-continuous process. The present disclosure extends to a method of producing the formulation, its use, and a method of producing porous polymer particles. [Background technology]

[0002] The properties of certain porous support particles to cause selective retardation based on either size or shape are well known. Such particles are used in chromatographic separation techniques, such as gel filtration, to separate biopolymers, such as proteins, DNA, RNA, polysaccharides, etc. Sieving particles are characterized by the presence of a microporous structure that exerts a selective effect on mobile solute macromolecules, restricting the passage of larger particles more than that of smaller particles. Thus, the usefulness of sieves lies in the properties of the particles to distinguish between molecules of various sizes and various shapes.

[0003] Affinity chromatography is a chromatographic method used to isolate proteins and other biological compounds. The technique is performed using an affinity ligand attached to a support particle and the resulting adsorbent packed into a chromatography column. The target protein is captured from the solution by selectively binding to the immobilized ligand. The bound protein can be washed to remove unwanted contaminants and then eluted in a highly pure form.

[0004] Successful separation using chromatographic techniques depends on particle size, particle size distribution, and particle porosity. The beads, once packed into the column, should be of high strength to support the liquid flow rates observed during purification and column regeneration. To ensure that any polymeric beads produced are suitable, batches can be tested to demonstrate the desired flow rates and / or to see that the beads have the desired porosity.

[0005] It has been found that the formulations used to manufacture the polymer beads can undergo physical changes over time. This means that beads produced later in a process can have a lower flow rate and / or higher porosity and may not be suitable for use in chromatography. To overcome this problem, when manufacturing the polymer beads, it has been necessary to only run small batches. This allows the entire formulation to be processed before degradation has a significant effect. However, it would be desirable to be able to process larger batch sizes, if possible, as this would allow for greater efficiency. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention results from the inventors' research, which seeks to overcome problems associated with the prior art. [Means for solving the problem]

[0007] According to a first aspect of the present disclosure, there is provided a formulation for the production of porous polymer particles, comprising: a polymer; a salt; and at least one buffer; wherein the salt is an antichaotropic salt, and the at least one buffer is present in a weight ratio of the at least one buffer to the polymer of at least 1:40.

[0008] Advantageously, the inventors have found that at least one buffer can stabilize the formulation. This means that it is possible to use large batches of the formulation of the first aspect to produce polymer beads with the required specifications so that they can be used in chromatography techniques. This allows these polymer beads to be produced more efficiently, which would not be possible using the formulations of the prior art. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 illustrates how flow rates in an agarose bead composition vary depending on where in the process the composition is manufactured. [Diagram 2] FIG. 1 illustrates how the porosity of an agarose bead composition varies depending on where in the process the composition is manufactured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Any weight ratio can be understood to be related to the weight ratio of pure components unless otherwise stated.Therefore, unless otherwise stated, weight ratio is not related to the weight of the solvent component of the solution containing each component.Furthermore, weight ratio does not incorporate the weight of any solvation molecule.For example, the weight ratio of at least one buffer to polymer can be understood to be the weight of pure buffer compared to the weight of pure polymer, and does not include the weight of any solvent that may be present in the formulation.

[0011] In some embodiments, the formulation comprises a solvent, which may comprise or consist of water.

[0012] The formulation may have a pH between 2.5 and 12 at 20° C., more preferably between 3 and 10, between 3.5 and 8, or between 4 and 6 at 20° C., and most preferably between 4.5 and 5.5 at 20° C. At least one buffer may be selected and provided in an appropriate concentration to maintain the formulation at this pH.

[0013] The at least one buffer may include or be a phosphate buffer, a sulfate buffer, a citrate buffer, an acetate buffer, a tris(hydroxymethyl)aminomethane (Tris) buffer, and / or a 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Preferably, the at least one buffer includes or is a phosphate buffer and / or a sulfate buffer. Most preferably, the at least one buffer includes or is a phosphate buffer.

[0014] The at least one buffer may comprise a cation and an anion. The anion may comprise dihydrogen phosphate, hydrogen phosphate, phosphate, pyrophosphate, sulfate, or hydrogen sulfate. Preferably, the buffer comprises dihydrogen phosphate, hydrogen phosphate, phosphate, or a combination thereof. The cation may comprise an alkali metal ion, an alkaline earth metal ion, a transition metal ion, or + NR4, where each R is independently hydrogen, alkyl, alkenyl, or alkynyl. Alkyl is C 1~6 Alkyl may be C 2~6 alkenyl, where alkynyl is C 2~6The cation may be an alkynyl. Thus, the cation may be a lithium ion, a sodium ion, a potassium ion, a beryllium ion, a magnesium ion, a calcium ion, an ammonium ion, or a trimethylammonium ion. Thus, the at least one buffer may comprise lithium dihydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, beryllium dihydrogen phosphate, magnesium dihydrogen phosphate, calcium dihydrogen phosphate, ammonium dihydrogen phosphate, dilithium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, beryllium hydrogen phosphate, magnesium hydrogen phosphate, ammonium hydrogen phosphate, lithium phosphate, sodium phosphate, potassium phosphate, beryllium phosphate, or ammonium phosphate.

[0015] In some embodiments, the at least one buffer comprises or is a combination of a dihydrogen phosphate buffer and a hydrogen phosphate buffer. Most preferably, the at least one buffer comprises or is composed of sodium dihydrogen phosphate and sodium hydrogen phosphate.

[0016] The weight ratio of dihydrogen phosphate to hydrogen phosphate may be at least 10:1, at least 20:1, at least 30:1, at least 35:1, at least 40:1, or at least 45:1. The weight ratio of dihydrogen phosphate to hydrogen phosphate may be less than 100:1, less than 80:1, less than 70:1, less than 60:1, less than 55:1, or less than 50:1. The weight ratio of dihydrogen phosphate to hydrogen phosphate may be between 10:1 and 100:1, between 20:1 and 80:1, between 30:1 and 70:1, between 35:1 and 60:1, between 40:1 and 55:1, or between 45:1 and 50:1.

[0017] The molar ratio of dihydrogen phosphate to hydrogen phosphate may be at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, or at least 55:1. The weight ratio of dihydrogen phosphate to hydrogen phosphate may be less than 200:1, less than 150:1, less than 100:1, less than 80:1, less than 65:1, or less than 60:1. The weight ratio of dihydrogen phosphate to hydrogen phosphate may be between 10:1 and 200:1, between 20:1 and 150:1, between 30:1 and 100:1, between 40:1 and 80:1, between 50:1 and 65:1, or between 55:1 and 60:1.

[0018] The weight ratio of the at least one buffer to the polymer may be less than 1:2, less than 1:3, less than 1:4, less than 1:5, less than 1:6, less than 1:6.5, or less than 1:7. The weight ratio of the at least one buffer to the polymer may be at least 1:30, at least 1:20, at least 1:15, at least 1:10, at least 1:8, or at least 1:7.5. The weight ratio of the at least one buffer to the polymer may be between 1:2 and 1:40, between 1:3 and 1:30, or between 1:4 and 1:20. The weight ratio of the at least one buffer to the polymer may be between 1:4 and 1:10, between 1:4 and 1:8, between 1:4 and 1:6, or between 1:4 and 1:5. The weight ratio of the at least one buffer to the polymer may be between 1:5 and 1:15, or between 1:6 and 1:10. In one embodiment, the weight ratio of the at least one buffer to the polymer may be between 1:6 and 1:8, or between 1:6 and 1:7. In a preferred embodiment, the weight ratio of the at least one buffer to the polymer may be between 1:6.5 and 1:8, or between 1:7 and 1:7.5. In an embodiment in which the at least one buffer comprises two or more different compounds, it may be appreciated that the weight ratio is calculated using the combined weight of the two or more different compounds.

[0019] In embodiments where the formulation includes a solvent, the at least one buffer may be present in an amount that is at least 3 g / L, at least 4 g / L, at least 5 g / L, at least 6 g / L, at least 7 g / L, at least 8 g / L, at least 9 g / L, or at least 10 g / L. The at least one buffer may be present in an amount that is less than 50 g / L, less than 45 g / L, less than 40 g / L, less than 35 g / L, less than 30 g / L, less than 25 g / L, less than 20 g / L, less than 15 g / L, less than 12.5 g / L, or less than 11 g / L. The at least one buffer may be present in an amount that is between 3 g / L and 50 g / L, between 4 g / L and 40 g / L, between 5 g / L and 30 g / L, between 6 g / L and 25 g / L, or between 7 g / L and 20 g / L. The at least one buffer may be present in an amount that is between 7g / L and 15g / L, between 7g / L and 10g / L, between 7.5g / L and 9g / L, or between 7.5g / L and 8.5g / L. The at least one buffer may be present in an amount that is between 8g / L and 15g / L, between 9g / L and 12.5g / L, between 10g / L and 11g / L, or between 10g / L and 12g / L. In embodiments where the at least one buffer comprises two or more different compounds, it may be appreciated that the concentration is calculated using the combined weight of the two or more different compounds.

[0020] In embodiments in which the formulation comprises a solvent, the at least one buffer may be present in an amount that is at least 0.001 M, at least 0.0025 M, at least 0.005 M, at least 0.01 M, at least 0.025 M, at least 0.05 M, at least 0.07 M, or at least 0.08 M. The at least one buffer may be present in an amount that is less than 2 M, less than 1 M, less than 0.5 M, less than 0.3 M, less than 0.2 M, less than 0.15 M, less than 0.1 M, or less than 0.09 M. The at least one buffer may be present in an amount that is between 0.001 M and 2 M, between 0.0025 M and 1 M, between 0.005 M and 0.5 M, between 0.01 M and 0.3 M, between 0.025 M and 0.2 M, between 0.05 M and 0.15 M, between 0.07 M and 0.1 M, or between 0.08 M and 0.09 M. In embodiments where the at least one buffer comprises two or more different compounds, it will be appreciated that the concentrations are calculated using the combined moles of the two or more different compounds.

[0021] In embodiments in which at least one buffer is provided as a solvate, when reciting a weight ratio or concentration, it can be recognized that the weight of the solvate molecules is not included.

[0022] The polymer may be a polysaccharide. The polysaccharide may be agarose.

[0023] In embodiments where the formulation includes a solvent, the polymer may be present in an amount that is at least 10 g / L, at least 20 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, at least 70 g / L, or at least 75 g / L. The polymer may be present in an amount that is less than 200 g / L, less than 175 g / L, less than 150 g / L, less than 125 g / L, less than 100 g / L, less than 90 g / L, less than 80 g / L, less than 77 g / L, less than 70 g / L, less than 65 g / L, or less than 60 g / L. The polymer may be present in an amount that is between 10 g / L and 200 g / L, between 20 g / L and 175 g / L, between 30 g / L and 150 g / L, between 40 g / L and 125 g / L, or between 50 g / L and 100 g / L. In one embodiment, the polymer may be present in an amount that is between 60 g / L and 90 g / L, between 70 g / L and 80 g / L, or between 75 g / L and 77 g / L. In another embodiment, the polymer may be present in an amount that is between 40 g / L and 80 g / L, between 45 g / L and 70 g / L, between 50 g / L and 60 g / L, or between 52 g / L and 57 g / L. The polymer may be present in an amount that is between 45 g / L and 55 g / L.

[0024] An antichaotropic salt may be understood to be a salt that will increase the hydrophobic effect in aqueous solution. Any salt that contains an anion to the left of the Hofmeister series may be considered to be an antichaotropic salt.

[0025] Thus, a salt may be considered to be an antichaotropic salt if it contains an anion selected from the left of the Hofmeister series, including, but not limited to, sulfate, phosphate, carbonate, citrate, acetate, and fluoride. In some embodiments, the antichaotropic salt is a sulfate or phosphate. Preferably, the antichaotropic salt is a sulfate.

[0026] It will be appreciated that the antichaotropic salt will include a cation. The cation may be an alkali metal ion, an alkaline earth metal ion, a transition metal ion, or a cation represented by the formula + NR4, where each R is independently hydrogen, alkyl, alkenyl, or alkynyl. Alkyl is C 1~6 Alkyl may be C 2~6 alkenyl, where alkynyl is C 2~6 The cation may be alkynyl. Thus, the cation may be lithium, sodium, potassium, beryllium, magnesium, calcium, ammonium, or trimethylammonium. Thus, the antichaotropic salt may be lithium sulfate, sodium sulfate, potassium sulfate, beryllium sulfate, magnesium sulfate, calcium sulfate, ammonium sulfate, trimethylammonium sulfate, lithium phosphate, sodium phosphate, potassium phosphate, beryllium phosphate, ammonium phosphate, or trimethylammonium phosphate. In some embodiments, the antichaotropic salt is ammonium sulfate.

[0027] The weight ratio of polymer to antichaotropic salt may be at least 0.1:1, at least 0.2:1, or at least 0.3:1. The weight ratio of polymer to antichaotropic salt may be at least 0.4:1, at least 0.5:1, at least 0.6:1, or at least 0.7:1, preferably at least 0.8:1, at least 0.85:1, at least 0.9:1, at least 0.95:1, or at least 1:1. The weight ratio of polymer to antichaotropic salt may be less than 2:1, less than 1.75:1, or less than 1.5:1, preferably less than 1.4:1, less than 1.3:1, less than 1.2:1, less than 1.1:1, or less than 1.05:1. The weight ratio of polymer to antichaotropic salt may be between 0.1:1 and 5:1, between 0.2:1 and 4:1, between 0.3:1 and 3:1, between 0.4:1 and 2:1, between 0.5:1 and 1.75:1, or between 0.6:1 and 1.5:1, preferably between 0.7:1 and 1.4:1, between 0.8:1 and 1.3:1, between 0.9:1 and 1.2:1, between 0.95:1 and 1.1:1, or between 1:1 and 1.05:1. The weight ratio of polymer to antichaotropic salt may be between 0.1:1 and 1:1, between 0.2:1 and 0.8:1, between 0.3:1 and 0.6:1, or between 0.4:1 and 0.5:1.

[0028] In embodiments where the formulation includes a solvent, the antichaotropic salt may be present in an amount that is at least 10 g / L, at least 20 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, at least 70 g / L, or at least 74 g / L. The antichaotropic salt may be present in an amount that is less than 200 g / L, less than 175 g / L, less than 150 g / L, less than 125 g / L, less than 100 g / L, less than 90 g / L, less than 80 g / L, or less than 75 g / L. The antichaotropic salt may be present in an amount that is between 10 g / L and 200 g / L, between 20 g / L and 175 g / L, between 30 g / L and 150 g / L, or between 40 g / L and 125 g / L. The anti-chaotropic salt may be present in an amount that is between 50 g / L and 100 g / L, between 60 g / L and 90 g / L, between 70 g / L and 80 g / L, or between 74 g / L and 75 g / L. The anti-chaotropic salt may be present in an amount that is between 60 g / L and 125 g / L, between 80 g / L and 120 g / L, between 100 g / L and 115 g / L, or between 110 g / L and 112 g / L.

[0029] In embodiments in which the formulation includes a solvent, the antichaotropic salt may be present in an amount that is at least 0.01 M, at least 0.05 M, at least 0.1 M, at least 0.2 M, at least 0.3 M, at least 0.4 M, at least 0.5 M, or at least 0.55 M. The antichaotropic salt may be present in an amount that is less than 2 M, less than 1.5 M, less than 1.2 M, less than 1 M, less than 0.8 M, less than 0.7 M, less than 0.6 M, or less than 0.57 M. The antichaotropic salt may be present in an amount that is between 0.01 M and 2 M, between 0.05 M and 1.5 M, between 0.1 M and 1.2 M, between 0.2 M and 1 M, between 0.3 M and 0.8 M, between 0.4 M and 0.7 M, between 0.5 M and 0.6 M, or between 0.55 M and 0.57 M.

[0030] Advantageously, the inventors have found that the addition of at least one buffer allows the amount of anti-chaotropic salt to be reduced, which also eliminates salting out of the polymer.

[0031] According to a second aspect, there is provided a method of producing a formulation for the production of porous polymer particles, comprising the steps of contacting a polymer, at least one buffer, and a salt, wherein the salt is an antichaotropic salt and the buffer is present in a weight ratio of the at least one buffer to the polymer of at least 1:40, thereby producing a formulation for the production of porous polymer particles.

[0032] The process of the second aspect preferably produces the formulation of the first aspect.

[0033] The step of contacting the polymer, at least one buffer, and an anti-chaotropic salt comprises: - contacting a polymer, at least one buffer, and a solvent to provide a first mixture; - dissolving the polymer in a solvent to provide a polymer solution; and - contacting the polymer solution with an anti-chaotropic salt may include:

[0034] The polymer, at least one buffer, anti-chaotropic salt and solvent may be as defined in relation to the first aspect.

[0035] The step of contacting the polymer, at least one buffer, and the solvent can include contacting the polymer with a buffer solution, the buffer solution comprising a solvent and at least one buffer. The buffer solution can be an aqueous solution. Thus, the solvent can be water.

[0036] Dissolving the polymer in the solvent to provide a polymer solution may include heating the first mixture to an elevated temperature. The elevated temperature may be at least 20° C., at least 30° C., at least 40° C., at least 50° C., at least 60° C., at least 70° C., at least 80° C., at least 90° C., at least 95° C., or at least 97° C. The elevated temperature may be between 20° C. and 500° C., between 30° C. and 400° C., between 40° C. and 300° C., between 50° C. and 200° C., between 60° C. and 175° C., between 70° C. and 150° C., between 80° C. and 125° C., between 90° C. and 110° C., between 95° C. and 100° C., or between 97° C. and 99° C.

[0037] The first mixture may be heated at the elevated temperature for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, or at least 30 minutes. The first mixture may be heated at the elevated temperature for between 5 and 300 minutes, between 10 and 120 minutes, between 15 and 60 minutes, between 20 and 45 minutes, or between 25 and 30 minutes.

[0038] The method may include cooling the polymer solution prior to contacting the polymer solution with an antichaotropic salt. The method may include cooling the polymer solution to a temperature below 95° C., below 90° C., below 85° C., below 80° C., below 75° C., or below 71° C. The method may include cooling the polymer solution to a temperature between 30° C. and 95° C., between 40° C. and 90° C., between 50° C. and 85° C., between 60° C. and 80° C., between 65° C. and 75° C., or between 69° C. and 71° C.

[0039] The step of contacting the polymer solution with an antichaotropic salt can include contacting the polymer solution with a salt solution, the salt solution comprising an antichaotropic salt and a solvent. The salt solution can be an aqueous solution. Thus, the solvent can be water. The salt solution can be at a temperature between 30°C and 95°C, between 40°C and 90°C, between 50°C and 85°C, between 60°C and 80°C, between 65°C and 75°C, or between 69°C and 71°C.

[0040] The volume ratio of polymer solution to salt solution can be between 1:1 and 20:1, between 2:1 and 15:1, between 3:1 and 10:1, between 4:1 and 8:1, between 4.5:1 and 7:1, between 5:1 and 6.5:1, between 5.25:1 and 6:1, or between 5.5:1 and 5.7:1.

[0041] It will be appreciated that at least one buffer, polymer and anti-chaotropic salt may be provided in suitable concentrations in the buffer solution, polymer solution and salt solution to result in the concentrations in the resulting formulation as defined in relation to the first aspect.

[0042] According to a third aspect there is provided the use of the formulation of the first aspect for producing porous polymer particles.

[0043] According to a fourth aspect, there is provided a method of producing porous polymer particles, the method comprising the step of supplying a formulation of the first aspect to an atomizer to produce porous polymer particles.

[0044] The method may include carrying out the method of the second aspect to produce the formulation before the step of feeding the formulation to the atomizer. The method of the second aspect may be completed in less than 10 hours before carrying out the method of the fourth aspect, more preferably in less than 8 hours, less than 6 hours, less than 4 hours, less than 2 hours, less than 1 hour, or less than 30 minutes before carrying out the method of the fourth aspect. As explained, the formulation of the first aspect degrades over time. Therefore, it is advantageous to carry out the method of the fourth aspect as soon as possible after the formulation is produced.

[0045] The formulation may be provided to the atomizer at a temperature of at least 20° C., at least 30° C., at least 40° C., at least 45° C., at least 50° C., at least 55° C., or at least 56° C. The formulation may be provided to the atomizer at a temperature of less than 100° C., less than 90° C., less than 80° C., less than 70° C., less than 65° C., less than 60° C., or less than 57° C. The formulation may be provided to the atomizer at a temperature of between 20° C. and 100° C., between 30° C. and 90° C., between 40° C. and 80° C., between 45° C. and 70° C., between 50° C. and 65° C., or between 53° C. and 61° C.

[0046] The method may include cooling the formulation prior to delivering the formulation to the atomizer. The method may include cooling the formulation at a rate of 5° C. / min or less, 2° C. / min or less, 1° C. / min or less, 0.5° C. / min or less, 0.2° C. / min or less, or 0.1° C. / min or less. The method may include cooling the formulation at a rate of between 0.0001° C. / min and 5° C. / min, between 0.0005° C. / min and 2° C. / min, between 0.001° C. / min and 1° C. / min, between 0.005° C. / min and 0.5° C. / min, between 0.01° C. / min and 0.2° C. / min, or between 0.05° C. / min and 0.1° C. / min.

[0047] The atomizer may be as described in US Pat. No. 7,207,499 B2.

[0048] The steps of feeding the formulation of the first embodiment into an atomizer to produce porous polymer particles include: - dispersing the formulation onto an atomization wheel; - rotating the spray wheel, thereby forming a layer of the formulation due to centrifugal force; - dividing the layer into filaments; and - flowing air over the wheel, thereby breaking up the filaments into porous polymer particles may include:

[0049] It can be appreciated that the speed of the atomizing wheel rotation can be selected according to the desired size of the porous polymer particles. Those skilled in the art can select a suitable rotation speed. In one embodiment, rotating the atomizing wheel can include rotating the wheel at a speed between 1000RPM and 25000RPM, between 2000RPM and 20000RPM, between 4000RPM and 15000RPM, between 4500RPM and 10000RPM, or between 5000RPM and 8000RPM.

[0050] Preferably, the method includes the step of transferring the polymer particles from the atomizer wheel to a tray.

[0051] In some embodiments, the method includes producing polymer particles, wherein at least 90%, at least 95%, or at least 97% of the particles have a diameter between 50 μm and 200 μm, between 60 μm and 150 μm, or between 76 μm and 141 μm.

[0052] As explained, the formulation of the first embodiment degrades over time. Thus, the length of the process time may depend on the specifications of the final product. The inventors have been able to obtain high quality porous particles after process times between 6 and 7 hours, but it should be recognized that longer process times may be used for lower specification products. Thus, the method may include continuously feeding the formulation of the first embodiment to the atomizer for at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours. The method may include continuously feeding the formulation of the first embodiment to the atomizer for less than 30 hours, less than 20 hours, less than 15 hours, less than 12 hours, less than 10 hours, less than 8 hours, or less than 7 hours. The method may comprise continuously supplying the formulation of the first aspect to the atomizer for between 30 minutes and 30 hours, between 1 hour and 20 hours, between 2 hours and 15 hours, between 3 hours and 12 hours, between 4 hours and 10 hours, between 5 hours and 8 hours, or between 6 hours and 7 hours.

[0053] The total volume of the formulation can vary depending on the process time, the desired polymer particle size, the diameter of the atomizing wheel and / or the number of atomizing wheels used. The inventors were able to obtain high quality porous particles with a diameter of about 100 μm using a 20 liter volume of the formulation. It can be appreciated that larger volumes can be used for products with larger bead sizes or lower specifications. Thus, the method may include continuously feeding at least 1 liter, at least 2 liters, at least 4 liters, at least 6 liters, at least 8 liters, at least 10 liters, at least 12 liters, at least 14 liters, at least 16 liters, at least 18 liters, or at least 20 liters of the formulation of the first embodiment to the atomizer. The method may include continuously feeding less than 60 liters, less than 50 liters, less than 45 liters, less than 40 liters, less than 35 liters, less than 30 liters, less than 28 liters, less than 26 liters, less than 24 liters, less than 22 liters, or less than 21 liters of the formulation of the first embodiment to the atomizer. The method may comprise the step of continuously supplying between 1 litre and 60 litres, between 2 litres and 50 litres, between 4 litres and 45 litres, between 6 litres and 40 litres, between 8 litres and 35 litres, between 10 litres and 30 litres, between 12 litres and 28 litres, between 14 litres and 26 litres, between 16 litres and 24 litres, between 18 litres and 22 litres, or between 19 litres and 20 litres of the formulation of the first aspect to the atomiser.

[0054] Every feature described in this specification (including any accompanying claims, abstract, and drawings), and / or every step of any disclosed method or process, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0055] For a more complete understanding of the present invention, and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which: FIG. 1 is a diagram showing how flow rates in an agarose bead composition vary depending on where in the process the composition is made; FIG. 2 is a diagram illustrating how the porosity of an agarose bead composition varies depending on where in the process the composition is made. EXAMPLES

[0056] Example 1 - Preparation of agarose beads using the first formulation Agarose bead compositions were prepared using the formulations shown in Table 1.

[0057] [Table 1]

[0058] Briefly, 85% water was combined with monobasic sodium phosphate. The agarose was then slowly poured into the water with vigorous mixing. This solution was heated to 97-99°C for 30 minutes and cooled to 70°C. A heating / cooling fluid was used in the reactor jacket to precisely control the temperature. The ammonium sulfate solution with the remaining water, maintained at 70°C, was added very slowly to the agarose solution with vigorous stirring. The final solution was cooled to 56-57°C at a rate of less than 0.1°C / min.

[0059] The formulation was then processed to produce a polymeric bead composition over a period of 300 minutes.

[0060] The flow rates of beads produced at different times during the procedure were analyzed to determine flow rate and porosity. Flow rates were measured using a 1.6 cm diameter column. The results are shown in Figures 1 and 2. Notably, as shown in Figure 1, the flow rate dropped off rapidly for beads produced later in the process. When the compositions produced throughout the process were combined, the resulting composition was still outside of applicant's specification requirements. Figure 2 also shows that the porosity of the beads increased during the process.

[0061] Example 2 - Production of agarose beads using a novel 7 liter batch formulation Agarose bead compositions were prepared using the novel formulations shown in Table 2.

[0062] [Table 2]

[0063] The formulation was prepared as described above. Specifically, the first step of the process involved combining 85% water with monobasic sodium phosphate and disodium phosphate. The other steps were the same.

[0064] It is noted that the new formulation eliminates salting out, a phenomenon that causes a foamy agarose cloud to form on the surface of the preparation solution, which was observed when prior art formulations were used. This is advantageous because it prevents the formation of trapped air in the formulation, which can affect surface area and bead density and reduce porosity.

[0065] Three different batches of the formulation were processed to produce polymer bead compositions. The resulting compositions were evaluated to ensure they met specifications, as shown in Table 3.

[0066] [Table 3]

[0067] It is noted that all of the batches showed improvements over the batches produced in Example 1. In particular, pressure vs. flow tests showed significantly higher ratios than those obtained when the compositions produced throughout the process of Example 1 were combined. It is noted that all of the batches met applicant's specification requirements.

[0068] Example 3 - Production of agarose beads using a novel 12 liter batch formulation Agarose beads were manufactured using the novel formulation shown in Table 4.

[0069] [Table 4]

[0070] The formulations were prepared using the methods described above.

[0071] Three different batches of the formulation were processed to produce polymer bead compositions. The resulting compositions were evaluated to ensure they met specifications, as shown in Table 5.

[0072] [Table 5]

[0073] Note that the pressure vs. flow test results changed because columns of different diameters were used. All of the batches met applicant's specification requirements.

[0074] Example 4 - Production of agarose beads using a new 15 liter batch formulation Agarose beads were manufactured using the novel formulation shown in Table 6.

[0075] [Table 6]

[0076] Formulations were prepared using the method described above, with a range of salt concentrations due to variations in the agarose.

[0077] Three different batches of the formulation were processed to produce polymeric bead compositions. The resulting compositions were evaluated to ensure they met specifications, as shown in Table 7.

[0078] [Table 7]

[0079] Again, all of the batches met applicant's specification requirements.

[0080] Example 5 - Investigating the effect of buffers on porosity Ammonium sulfate is known to affect the porosity of the resulting composition, but as shown above, the inventors have been able to reduce the ammonium sulfate concentration while still maintaining an acceptable level of porosity.

[0081] The monosodium phosphate and di-sodium phosphate in the above examples were added as buffers to stabilize the formulations. The inventors decided to test if other buffers could be used and if this could affect the porosity of the resulting composition.

[0082] The phosphate buffer described in Examples 2 through 4 provides a formulation with a pH of about 5 at 20° C. This was replaced with a 5 mM acetate buffer to provide a formulation with a pH of 5.0 at 20° C. The acetate formulation was processed to produce a polymer bead composition and evaluated for porosity. The results are shown in Table 8.

[0083] [Table 8]

[0084] The above table shows that the buffer affects the porosity of the composition.

[0085] Example 6 - Further investigating the effect of buffer on porosity We then investigated the effect of using additional alternative buffers, the results of which are shown in the table below.

[0086] [Table 9]

[0087] It should be noted that pH did not appear to affect the properties of the composition, however, phosphate buffer significantly increased the porosity of the composition compared to other buffers.

[0088] Example 7 - Investigating the effect of salt choice on porosity The inventors decided to investigate the effect of the choice of salt on the porosity of the resulting composition.

[0089] First, we investigated the effect that sulfate groups had by preparing polymer beads using formulations containing either ammonium sulfate or ammonium chloride. The ammonium concentration was kept constant for the two formulations, and the results are shown in Table 10.

[0090] [Table 10]

[0091] We then looked to see whether replacing the ammonium group would have an effect.

[0092] [Table 11]

[0093] Table 10 shows that when ammonium sulfate is replaced by ammonium chloride, the porosity is significantly reduced. On the other hand, Table 11 shows that sodium chloride produces almost no porosity, which is consistent with the results observed with ammonium chloride. Sodium sulfate produces about two-thirds of the porosity observed with ammonium sulfate, but the difference may be due to the difference in concentration rather than the replacement of ammonium ions. Hence, the table shows that it is possible to induce porosity when ammonium ions are replaced. Thus, it can be seen that the choice of anion is more important to induce porosity.

[0094] Example 8 - Testing further compositions The inventors then produced further formulations, which are set out in the table below.

[0095] [Table 12]

[0096] The formulations were produced using the methods described above. A batch of Formulation A was used to produce a polymeric bead composition having a desired diameter of 100 μm, a batch of Formulation B was used to produce polymeric bead compositions having desired diameters of 60 μm and 100 μm, and a batch of Formulation C was used to produce a polymeric bead composition having a desired diameter of 200 μm. The resulting compositions were evaluated to ensure that they met specifications, as shown in Table 13.

[0097] [Table 13]

[0098] It is noted that due to the lower agarose and higher salt concentrations, these compositions had higher porosity and lower flow rates. Thus, the formulation can be varied, if necessary, to fine-tune the properties of the resulting composition.

[0099] method All of the polymeric bead compositions described in the examples were produced using the apparatus and methods described in US Pat. No. 7,207,499 B2, which is incorporated by reference.

[0100] Pressure vs Flow Analysis Pressure versus flow for agarose beads made from the formulations described in the Examples was measured in two different ways: by maintaining a constant flow through a column of beads and measuring the pressure, or by maintaining a constant pressure and measuring the flow.

[0101] Method 1 (constant flow) This method was used in the pressure versus flow analysis reported in Example 2.

[0102] The agarose beads were washed a total of four times using purified water, a final aliquot of water was added and allowed to drain under gravity. 66 g of settled beads were weighed and made up to 80 g with purified water, then degassed for 10 minutes.

[0103] The suspension was transferred to a 1.6 cm diameter column and equilibrated for 90 min at a flow rate of 1.0 mL / min. After equilibration, a bed height in the range 30±1 cm was expected. The pressure was measured for 10 min at a flow rate of 0.8 mL / min, and if the pressure was stable for 10 min, the flow rate was increased by 0.2 mL / min and the pressure measurement was repeated. Further increases in flow and further pressure measurements were continued until the stopped pressure was stable for 10 min. If a stable pressure could not be obtained at 0.8 mL / min, the flow was reduced to 0.7 m / min and the pressure measurement was repeated.

[0104] For each set of measurements, the linear flow rate was calculated using the following formula:

[0105]

number

[0106] where 2.01 is the cross-sectional area of ​​the column (cm 2 ) was.

[0107] The maximum linear flow rate obtained was recorded.

[0108] Method 2 (Constant Pressure) This method was used in the pressure versus flow analyses reported in Examples 3 and 4.

[0109] The agarose beads were washed a total of four times using purified water, a final aliquot of water was added and allowed to drain under gravity. 142 g of settled beads were weighed and made up to 180 g with purified water, then degassed for 15 minutes.

[0110] The suspension was transferred to a column with a diameter of 3.2 cm and equilibrated at a pressure of 10 psi for 20 minutes. After equilibration, a bed height in the range 15±1 cm was expected. The pressure was adjusted to 2.5 psi and allowed to stabilize for 5 minutes, and solute from the column was collected over the next 5 minutes and the volume was measured. The analysis was repeated at pressures of 5.0 psi, 7.5 psi, 10.0 psi, 12.5 psi, and 15.0 psi. The actual flow rate for each pressure was calculated using the following formula:

[0111]

number

[0112] where 5 was the fraction in which the volume was recovered.

[0113] For each pressure level, the linear flow rate was calculated using the following formula:

[0114]

number

[0115] where 8.04 is the cross-sectional area of ​​the column (cm 2 ) was.

[0116] The maximum linear flow rates obtained were reported as well as the corresponding bed heights and pressures.

[0117] Maximum linear flow results were normalized to a bed height of 15 cm using the following formula: Normalized flow rate = linear flow rate (cm / hour) x F where F is the measured gel bed height divided by 15 and is expected to range from 0.933 to 1.067.

[0118] Bead size analysis Bead size was measured using a benchtop FlowCam using the instrument's standard procedures.

[0119] Porosity analysis Porosity was measured using a Waters HPLC system (solvent selector, pump 1515, autosampler 717 plus, column selector, and UV-VIS detector 2487). A Waters AP-2 2.0 cm (ID) x 20.0 cm (L) graduated glass column equipped with an adapter (piston and column bottom) and a packing funnel was filled with a pH 7.5 Tris / HCl buffer (50 mM) KCl (100 mM) solution and degassed agarose beads made from the formulations described in the examples. The column was allowed to pack for 60 min. The compressed gel bed height was confirmed to be at least 16 cm, and the HPLC system was allowed to equilibrate under analytical conditions (flow rate 0.70 ml / min, detector wavelength 280 nm, and autosampler temperature 4°C). The system was allowed to equilibrate for 45 min or until a stable baseline integration line was obtained.

[0120] A solution of blue dextran (2 mg / ml) and a further solution of thyroglobulin (8 mg / ml) were made up in buffer (Tris / HCl (50 mM) KCl (100 mM) pH=7.50).

[0121] The gel bed height was noted and 250 μL of blue dextran solution was injected. The porosity for dextran blue (KAV) was calculated as follows:

[0122]

number

[0123] During the ceremony: Tr (チログロブリン) is the retention time of thyroglobulin; Tr (デキストランブルー) is the retention time of dextran blue; πcm 2 is the cross-sectional area of ​​the column.

Claims

1. 1. A formulation for producing porous polymer particles, comprising: a polymer; a salt; and at least one buffer; wherein the polymer is a polysaccharide, the salt is an antichaotropic salt, and the at least one buffer is present in a weight ratio of the at least one buffer to the polymer of at least 1:

40.

2. 10. The formulation of claim 1, comprising a solvent, said solvent being water.

3. the polymer is present in an amount between 10 g / L and 200 g / L, between 20 g / L and 175 g / L, between 30 g / L and 150 g / L, between 40 g / L and 125 g / L, or between 50 g / L and 100 g / L; or 3. The formulation of claim 2, wherein the antichaotropic salt is present in an amount that is between 0.01M and 2M, between 0.05M and 1.5M, between 0.1M and 1.2M, between 0.2M and 1M, between 0.3M and 0.8M, between 0.4M and 0.7M, between 0.5M and 0.6M, or between 0.55M and 0.57M.

4. 3. The formulation of claim 1 or 2, wherein the at least one buffer comprises or is a phosphate buffer, a sulfate buffer, a citrate buffer, an acetate buffer, a tris(hydroxymethyl)aminomethane (Tris) buffer, and / or a 2-(N-morpholino)ethanesulfonic acid (MES) buffer, wherein the at least one buffer comprises or may be a phosphate buffer, and wherein the at least one buffer comprises or may be a combination of dihydrogen phosphate and hydrogen phosphate.

5. 3. The formulation of claim 1 or 2, wherein the weight ratio of the at least one buffer to the polymer is between 1:2 and 1:40, between 1:3 and 1:30, between 1:4 and 1:20, between 1:5 and 1:15, between 1:6 and 1:10, between 1:6.5 and 1:8, or between 1:7 and 1:7.

5.

6. 3. The formulation of claim 1 or 2, having a pH at 20°C of between 2.5 and 12, between 3 and 10, between 3.5 and 8, between 4 and 6, or between 4.5 and 5.

5.

7. The composition of claim 1 or 2, wherein the polysaccharide is agarose.

8. 3. The formulation of claim 1 or 2, wherein the antichaotropic salt is a salt comprising an anion selected from the group consisting of sulfate ions, phosphate ions, carbonate ions, citrate ions, and fluoride ions, and wherein the antichaotropic salt may be a sulfate salt.

9. 3. The formulation of claim 1 or 2, wherein the weight ratio of the polymer to the antichaotropic salt is between 0.5:1 and 2:1, between 0.6:1 and 1.75:1, or between 0.7:1 and 1.5:1, preferably between 0.8:1 and 1.4:1, between 0.85:1 and 1.3:1, between 0.9:1 and 1.2:1, between 0.95:1 and 1.1:1, or between 1:1 and 1.05:

1.

10. The formulation of claim 1 or 2, wherein the salt and the at least one buffer are chemically different.

11. 1. A method of producing a formulation for the production of porous polymer particles, comprising the step of contacting a polymer, at least one buffer, and a salt, wherein the polymer is a polysaccharide and the salt is an antichaotropic salt, and the at least one buffer is present in a weight ratio of the at least one buffer to the polymer of at least 1:40, thereby producing the formulation for the production of porous polymer particles.

12. contacting the polymer, the at least one buffer, and the salt; - contacting the polymer, the at least one buffer, and a solvent to provide a first mixture; - dissolving the polymer in the solvent to obtain a polymer solution; and - contacting said polymer solution with said anti-chaotropic salt wherein dissolving the polymer in the solvent to provide a polymer solution may comprise heating the first mixture to an elevated temperature, wherein the elevated temperature is between 20°C and 500°C, between 30°C and 400°C, between 40°C and 300°C, between 50°C and 200°C, between 60°C and 175°C, between 70°C and 150°C, between 80°C and 125°C, between 90°C and 110°C.

12. The method of claim 11, wherein the first solution is cooled to a temperature between 30°C and 95°C, between 40°C and 90°C, between 50°C and 85°C, between 60°C and 80°C, between 65°C and 75°C, or between 69°C and 71°C, and optionally includes cooling the first solution prior to contacting the first solution with the salt, wherein the first solution is cooled to a temperature between 30°C and 95°C, between 40°C and 90°C, between 50°C and 85°C, between 60°C and 80°C, between 65°C and 75°C, or between 69°C and 71°C.

13. 3. Use of the formulation according to claim 1 or 2 for producing porous polymer particles.

14. 10. A method for producing porous polymer particles, comprising the step of feeding the formulation of claim 1 or 2 into an atomizer to produce porous polymer particles.

15. and / or carrying out the method of claim 11 or 12 to produce the formulation before the step of feeding the formulation to the atomizer. continuously feeding the formulation into a nebulizer for at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours; and / or 15. The method of claim 14, comprising continuously supplying at least 1 liter, at least 2 liters, at least 4 liters, at least 6 liters, at least 8 liters, at least 10 liters, at least 12 liters, at least 14 liters, at least 16 liters, at least 18 liters, or at least 20 liters of the formulation to the atomizer.

Images