Process for the preparation of polymer-filled chromatography resins

Abstract

Methods for preparing polymer-filled chromatographic resins and their applications are provided.

Description

Background Technology

[0001] Purifying viruses, proteins bound to large particles, and other biomacromolecules from smaller impurities currently involves the use of various separation methods, including but not limited to size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and / or centrifugation. SEC requires large columns packed with expensive size exclusion resins, low flow rates, and limited sample loading volumes. IEX and HIC have limited selectivity. Centrifugation can only be applied to large biomolecules, where the medium is relatively dense compared to the medium in which the biomolecules are suspended. Summary of the Invention

[0002] A method for preparing a chromatographic resin comprising an insoluble porous polymer is provided. In some embodiments, the method includes adding the chromatographic resin to a solution containing a molten polymer while stirring to allow the molten polymer to absorb or otherwise enter the pores of the chromatographic resin. The solution is then atomized, and insoluble porous polymer-filled chromatographic resin beads are collected in a water bath. Excess polymer is then removed from the insoluble porous polymer-filled chromatographic resin beads by, for example, passing the insoluble porous polymer-filled chromatographic resin beads through a sieve having openings of about 50 μm to about 200 μm and / or washing the insoluble porous polymer-filled chromatographic resin beads. In some embodiments, the temperature of the molten polymer solution is about 100°C. In some embodiments, the polymer is agarose, and the concentration of agarose ranges from about 0.5% to about 8%. In some embodiments, the chromatographic resin is a macroporous resin. In some embodiments, the chromatographic resin is hydroxyapatite microspheres. In some embodiments, the macroporous resin comprises acrylamide, methacrylate, polystyrene, or silica. In some embodiments, the macroporous resin comprises a copolymer of 3-allyloxy-1,2-propanediol and vinylpyrrolidone crosslinked with N,N'-methylenebisacrylamide. In some embodiments, the macroporous resin comprises glycidyl methacrylate and diethylene glycol dimethacrylate. In some embodiments, atomizing the solution comprises surrounding the solution ejected from the nozzle with an airflow (e.g., from about 20°C to about 90°C, at a velocity of about 15 L / min to about 25 L / min). In some embodiments, the water bath temperature is from about 4°C to about 40°C.

[0003] A method for chromatography using a polymer-filled chromatographic resin is also provided. In some embodiments, the method includes contacting a sample containing a target molecule with an insoluble porous polymer-filled chromatographic resin under conditions that prevent the target from being captured by the chromatographic resin, and collecting the target molecule from the insoluble porous polymer-filled chromatographic resin. In some embodiments, the sample contains contaminants captured by the chromatographic resin. In some embodiments, the target molecule is a protein-nanoparticle conjugate, and the contaminant is a free (uncoupled) protein. In some embodiments, the sample contains a protein-nanoparticle conjugate, a free protein, and a buffer solution. In some embodiments, the target is a virus. In some embodiments, the sample further contains a surfactant (e.g., a polyalkylene glycol or Pluronic F-68). In some embodiments, the collection step includes collecting one or more fractions rich in the target molecule from the insoluble porous polymer-filled chromatographic resin. In some embodiments, the collection step includes applying centrifugation or vacuum to the insoluble porous polymer-filled chromatographic resin and collecting one or more fractions rich in the target molecule from the insoluble porous polymer-filled chromatographic resin. In some embodiments, the protein is an antibody. In some embodiments, the protein is an IgG antibody. In some embodiments, the protein-nanoparticle conjugate is a protein-polymer dot conjugate. In some embodiments, the sample further comprises free nanoparticles, and the insoluble porous polymer-filled chromatographic resin separates the free nanoparticles from the conjugate.

[0004] Brief description of the attached figures

[0005] Figure 1A Images showing agarose-filled hydroxyapatite microspheres.

[0006] Figure 1B Images of stained agarose-hydroxyapatite microspheres after dispersing hydroxyapatite in HAc-NaAc buffer at pH 3.5 are shown.

[0007] Figure 2 An image of an agarose-filled UNOsphere is shown. Detailed Implementation

[0008] The inventors have discovered a novel method for preparing chromatographic resins filled with insoluble porous polymers exhibiting multimodal properties (i.e., size exclusion and trapping modes). The inventors have also discovered that insoluble porous polymers can be introduced into the resin without significantly affecting its selectivity or binding capacity. The scalable method does not use organic solvents and is therefore environmentally friendly. This method produces separate polymer-filled chromatographic resin beads, without clusters of beads within the polymer.

[0009] definition

[0010] The term "hydroxyapatite" refers to the structure with the formula Ca. 10 Hydroxyapatite is an insoluble calcium phosphate hydroxylated mineral (PO4)6(OH)2. Hydroxyapatite chromatographic resins are considered multimode resins due to their multiple interaction modes with biomolecules. The primary interaction modes are phosphoryl cation exchange and calcium metal affinity. Hydroxyapatite is commercially available in various forms, including but not limited to, hydroxyapatite microspheres, which are chemically pure forms of hydroxyapatite sintered at high temperatures to modify them from a crystalline form to a ceramic form. Hydroxyapatite microspheres are spherical with particle sizes ranging from about 10 micrometers to about 100 micrometers, typically with nominal diameters of 20 micrometers, 40 micrometers, and 80 micrometers. Hydroxyapatite microspheres (or HAM) are macroporous and come in three types: Type I, with medium porosity and relatively high binding capacity; Type II, with even greater porosity and lower binding capacity; and Type XT, with medium porosity, relatively high binding capacity, and excellent pressure flow properties. All apatite-based resins in this paragraph are available from Bio-Rad Laboratories, Inc. (Hercules, California, USA).

[0011] The term "antibody" refers to an immunoglobulin or a fragment thereof. This term includes, but is not limited to, polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG, and IgM classes derived from human or other mammalian cell lines, including natural or genetically modified forms such as humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, transferred, and in vitro generated antibodies. "Antibody" includes complex forms, including but not limited to fusion proteins containing immunoglobulin moieties. "Antibody" also includes antibody fragments such as Fab, F(ab′)2, Fv, scFv, Fd, dAb, Fc, and other combinations, whether or not they retain antigen-binding function.

[0012] The term "protein" can be used to refer to an amino acid polymer or a group of two or more amino acid polymers that interact or bind together. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.

[0013] The term "sample" refers to any composition containing a target molecule that needs to be purified. In some embodiments, the target molecule to be purified is a protein-nanoparticle conjugate (e.g., an antibody-nanoparticle conjugate) or a virus.

[0014] The term "contaminant" refers to any impurities to be removed from a sample. In some embodiments, the sample is a coupling reaction mixture of antibody-nanoparticle conjugate and unreacted components, and the contaminant is uncoupled (unreacted or free) antibody (and optionally uncoupled nanoparticles).

[0015] As used herein, the terms “an,” “a,” and “the” are intended to mean “one or more.” As used herein, the term “about” refers to the listed number and any value within 10% of the listed number. Thus, “about 5” means any value between 4.5 and 5.5, inclusive.

[0016] Method for preparing polymer-filled chromatographic resins

[0017] In some embodiments, the method of preparing a polymer-filled chromatographic resin includes adding the chromatographic resin to a solution containing molten polymer while stirring to allow the molten polymer to absorb or otherwise enter the pores of the chromatographic resin. In embodiments, the polymer is dispersed in water heated to a temperature above the polymer's melting point. In one embodiment, the polymer is insoluble such that the aqueous conditions of chromatography do not remove the polymer from the chromatographic resin during the chromatographic process. In embodiments, the polymer is porous enough that filling the chromatographic resin does not prevent macromolecules (e.g., proteins, nucleic acids, etc.) from interacting with sites within the chromatographic resin, thereby substantially maintaining the selectivity and binding capacity of the chromatographic resin. The chromatographic resin is porous and allows target molecules to interact with the chromatographic resin at least partially through the pores in the resin. Therefore, the polymer should not significantly interfere with this interaction.

[0018] Various polymers can be used to pack chromatographic resins. In some embodiments, the polymer is agarose. Agarose (e.g., 0.5-8%) can be introduced into the chromatographic resin by heating the agarose to melt it and mixing the molten agarose solution with the chromatographic resin. In some embodiments, the agarose solution is heated to about 100°C. In some embodiments, the agarose solution is heated from about 60°C to about 100°C.

[0019] The molten polymer / chromatographic resin solution is then atomized by flowing the solution through a nozzle and surrounding the solution ejected from the nozzle with an airflow to form chromatographic resin beads in which the polymer is coated on the outer surface of the beads and within the pores of the beads, and viscous polymer droplets. In some embodiments, the nozzle is a binary nozzle, for example, in which combined hot air and suspension flow to the nozzle opening. In some embodiments, the temperature of the airflow is from about 20°C to about 90°C. In some embodiments, the airflow velocity is from about 15 L / min to about 25 L / min (e.g., about 20 L / min). To prevent recombination of the resin beads and polymer droplets, the resin beads and polymer droplets are collected in a water bath immediately after atomization. The polymer droplets and the polymer incorporated within the resin pores and on the resin surface solidify. The polymer-filled resin beads are collected at the bottom of the water bath, while excess polymer (i.e., solid polymer droplets) remains suspended in the water. Excess polymer is removed from the insoluble porous polymer-filled chromatographic resin beads, for example, by passing the beads suspended in water through a sieve with openings ranging from about 50 μm to about 200 μm. In some embodiments, 120 μm and 65 μm sieves are used to remove excess polymer. Alternatively, excess polymer is removed by washing polymer-filled resin beads. In some embodiments, excess polymer is removed by decanting the suspension. In some embodiments, the nozzle diameter is 1-3 mm, for example, 2 mm. In some embodiments, the water bath temperature is from about 4°C to about 40°C.

[0020] In some embodiments, the agarose concentration in the heated agarose solution (e.g., greater than about 0.5%, 1%, 2%, 3%, or 4% agarose) is sufficient to fill the pores of the chromatographic resin and coat the outside of the resin. The agarose solution inside the beads forms an insoluble gel at temperatures below about 40°C.

[0021] In some embodiments, agarose is not cross-linked. For example, as described in the examples, agarose can be introduced into the chromatographic resin in a non-cross-linked form. However, in some embodiments, a cross-linking agent (e.g., a chemical cross-linking agent, including but not limited to, divinyl sulfone, epichlorohydrin, butanediol diglycidyl ether, 1,3-dichloropropanol, 2,3-dibromopropanol) is introduced after the agarose has been filled into the chromatographic resin. Therefore, in some embodiments, the agarose within the chromatographic resin will be cross-linked.

[0022] Typically, agarose is not functionalized. Therefore, in some embodiments, agarose is not modified to interact with the components of the sample during chromatography, thereby providing substantially the same selectivity as polymer-free chromatographic resins.

[0023] A variety of macroporous resins suitable for chromatography can be used in the methods described herein. In some embodiments, the chromatographic resin is ceramic hydroxyapatite. In some embodiments, type I or type II ceramic hydroxyapatite can be used (i.e., any porosity can be used). In some embodiments, the resin is functionalized with, for example, ion-exchange functional groups, hydrophobic interaction functional groups, mixed-mode functional groups, or affinity ligands. The optimal porosity for any particular protein separation or purification will vary depending on the composition of the protein or source mixture. In some embodiments, the chromatographic resin is a macroporous resin comprising acrylamide, methacrylate, polystyrene, or silica. In one embodiment, the macroporous resin comprises a copolymer of 3-allyloxy-1,2-propanediol and vinylpyrrolidone crosslinked with N,N'-methylenebisacrylamide. In another embodiment, the macroporous resin comprises glycidyl methacrylate and diethylene glycol dimethacrylate. Examples of such macroporous resins include, but are not limited to: UNOsphere Q, UNOsphere S, UNOsphere Rapid S, Macro-Prep High Q, Macro-Prep DEAE, Macro-Prep 25 Q, Macro-Prep CM, Macro-Prep 25 S, Nuvia S, and Nuvia Q (all from Bio-Rad). Other examples of macroporous resins include, but are not limited to: Toyopearl SP, CM Q, DEAE, DGigaCap S, CM, and Q from Tosoh Bioscience; Poros HS, XS, HQ, and XQ from Thermo Fisher Scientific; Eshmuno S, Q, CPX, CPS, and HCX from Millipore; and Sepharose Q, Sepharose S, Capto Q, Capto S, Capto MMC, and CaptoAdhere from GE Healthcare.

[0024] Polymer-packed resins can be used as chromatographic solid phases in the form of packed beds and can constitute the entire packed bed or a major portion of it, for example, 50% or more of its volume. The packed bed can be held in a container of any construction, and purification, cleaning, and regeneration within the resin can be performed as batch processes, continuous processes, or a hybrid batch / continuous process. In one embodiment, the container is a column with a suitable length relative to its width, and suitable processes include continuous processes, such as continuous flow through the column.

[0025] Chromatographic resins (e.g., chromatographic beads) can have size exclusion and capture modes. Size exclusion modes separate molecules, complexes, or particles based on their size or molecular weight. The beads have pore sizes such that molecules, complexes, or particles exceeding a size threshold are excluded from the pores and collected in the void volume, exclusion volume, or column permeate. Smaller proteins and other molecules can enter the pores of the beads and be captured by them. As used herein, the molecular weight cutoff size of a bead refers to the maximum size of a protein or molecule that can enter the pore. As the percentage of agarose in the pores filled with hydroxyapatite microspheres increases, the pores become smaller, resulting in a decrease in the molecular weight cutoff size. Therefore, the molecular weight cutoff value of beads filled with 4% agarose will be less than that of beads filled with 0.5% agarose.

[0026] Method using polymer-filled chromatographic resins

[0027] The polymer-filled chromatographic resins described herein can be used in chromatographic methods. In one embodiment, the method includes contacting a sample containing target molecules with a polymer-filled chromatographic resin (e.g., a plurality of agarose-filled hydroxyapatite microspheres) under conditions that prevent the target from being trapped by the beads. In one embodiment, the sample contains contaminants trapped by the polymer-filled chromatographic resin.

[0028] Before applying the sample to the polymer-packed chromatographic resin, the resin is typically equilibrated in a buffer or salt used to load the sample. Typically, the same conditions and reagents as in standard chromatography are used. For example, for chromatography based on ceramic hydroxyapatite, any of a variety of buffers or salts can be used, including those with cations such as sodium, potassium, ammonium, magnesium, and calcium, and anions such as chlorine, fluorine, acetic acid, phosphoric acid, and citric acid. The pH of the equilibration solution is typically about 6.0 or higher, and in many cases, the pH ranges from about 6.5 to about 8.6 or from about 6.5 to about 7.8. In some embodiments, equilibration can occur in a solution containing Tris or sodium phosphate buffer. The sodium phosphate buffer can be present, for example, at a concentration of about 0.5 mM to about 50 mM, or about 10 mM to about 35 mM.

[0029] As described above, the chromatographic steps described herein can be performed in conventional purification configurations, including but not limited to packed columns and fluidized bed or expanded bed columns, and can be performed by any conventional chromatographic method, including batch modes of loading, washing, and elution, as well as continuous or flow-through modes. In some embodiments, the medium is packed in columns with diameters ranging from less than 0.5 cm to greater than 1 m and column heights ranging from less than 1 cm to greater than 30 cm. In one embodiment, the resin is provided in a centrifugal column. The sample is applied to the top of the centrifugal column and then forced through the column by centrifugation or vacuum. In some cases, the resin is provided in the chromatographic column, and the sample is applied to the top of the column and forced through the column by gravity. The column can be run from top to bottom or bottom to top under pressure or without pressure, and the flow direction of the fluid within the column can be reversed during this process. In some cases, it may be advantageous to reverse the liquid flow while maintaining the packed bed configuration.

[0030] The methods described herein can be used to purify various types of target molecules, including viruses, naturally occurring proteins, and recombinant proteins. In some embodiments, the target molecule is conjugated or attached to a reporter, such as nanoparticles. In some embodiments, the target molecule is an antibody (e.g., IgG) conjugated to nanoparticles. Nanoparticles are nanoscale particles, for example, from about 1 nm to about 1000 nm. In some embodiments, the particles are between 1-300 nm, 5-500 nm, or 10-50 nm. Many nanoparticles are approximately spherical in shape, which makes the size the radius or diameter of the spherical particle. The hydrodynamic radius or diameter can also be used to define the nanoparticle size.

[0031] In some embodiments, the nanoparticles are fluorescent semiconductor polymer dots (pdots). Examples of such dots are described, for example, in Wu, C., et al., Chem. Mater. 21: 3816-3822 (2009); Rahim, NAA, et al., Adv. Mater. 21: 3492-3496 (2009); Rong et al., ACS Nano 7(1): 376-84 (2013); patent publications US 2013 / 0266957; WO2012 / 054525; and US 2012 / 0282632. Colorimetric pdots can be produced by collapsing a polymer into stable submicron-sized particles. The pdot nanoparticles provided herein can be formed by any method known in the art for collapsing polymers, including but not limited to precipitation-dependent methods, emulsion-dependent methods (e.g., microemulsions or microemulsions)-dependent methods, and condensation-dependent methods. The size of the PDOT nanoparticles depends on the molecular weight of the polymer used to generate the PDOT (see, for example, Zhang, Y., et al., Chem Sci. 6(3): 2102-2109 (2015) and U.S. Patent 9,382,473). In some embodiments, the molecular weight of the individual PDOTs ranges from about 500,000 Daltons to about 15,000,000 Daltons, or from about 1,800,000 Daltons to about 7,000,000 Daltons.

[0032] Other exemplary nanoparticles that can be used in the methods described herein include, but are not limited to, magnetic nanoparticles, quantum dots, and gold nanoparticles. Magnetic nanoparticles are a class of nanoparticles that can be manipulated using a magnetic field gradient. Magnetic nanoparticles are formed from magnetic or paramagnetic elements, including but not limited to iron, nickel, and cobalt and their compounds. Quantum dots are nanoparticles formed from inorganic semiconductor materials. Gold nanoparticles (e.g., colloidal gold) have optical properties that are advantageous for biomedical applications and are described, for example, Huang, X., et al., Journal of Advanced Research 1(1): 13-28 (2010).

[0033] Nanoparticles can be functionalized as needed to attach them to proteins. Exemplary functionalizations of nanoparticles are described in the aforementioned U.S. Patent Publication No. 2012 / 0282632. For example, nanoparticles can be functionalized to present one or more carboxylic acid moieties, which can then be used to attach one or more linkers to proteins. The conjugate components (e.g., proteins and nanoparticles) can be covalently or non-covalently linked. An example of a non-covalent link is a biotin-streptavidin affinity linker, in which one member of the conjugate is biotinylated while another member of the conjugate is linked to streptavidin. Other examples of coupling options include, but are not limited to, direct coupling of nanoparticles to protein amines; modifying nanoparticles with maleimide and subsequently coupling them to proteins with exposed thiol groups (e.g., by treating proteins with mercaptoethylamine or 2-iminothiolane (Traut's reagent)); modifying nanoparticles with hydrazine and coupling them to proteins with oxidized polysaccharides (aldehydes); or using click chemistry (e.g., modifying nanoparticles with strained alkyne and coupling them to proteins modified with azides).

[0034] Any type of coupling method can be used to couple proteins to nanoparticles. Typically, an excess of protein is provided in the coupling reaction to produce the desired yield of the conjugate. This results in a large amount of free (uncoupled) protein after the coupling reaction. In some embodiments, a certain amount of free uncoupled nanoparticles is also present in the reaction mixture. The methods described herein can be used to purify the conjugate from the free uncoupled member of the coupling reaction. In some embodiments, a reagent is applied that will react with the remaining reactive groups and prevent further reactions. For example, coupling between maleimide-functionalized nanoparticles and thiolated or reduced proteins will be stopped or quenched with an alkylating agent, including but not limited to N-ethylmaleimide. Reactions between NHS-added nanoparticles and proteins will be stopped or quenched with amines, including but not limited to ethanolamine.

[0035] Once coupling has been performed, the resulting conjugate mixture (e.g., nanoparticle / protein conjugate, unreacted free protein, and optionally free nanoparticles) is adjusted to establish appropriate pH, conductivity, and / or salt concentration. The conjugate mixture (i.e., the sample to be purified) can be adjusted, for example, by exchanging the conjugate buffer with a chromatography resin equilibration buffer. Exemplary buffer compounds include, but are not limited to, phosphates, HEPES, MES, and Tris. In some embodiments, the equilibration buffer contains about 10 mM to about 30 mM (e.g., 10 mM, 20 mM, or 30 mM) of HEPES. In some embodiments, the equilibration buffer contains about 5 mM to about 50 mM (e.g., 5 mM, 10 mM, 25 mM) of phosphate (PO42-).3- In some embodiments, the pH range of the equilibration buffer is from about 5 to about 8 (e.g., about 6, about 7, or about 8). In some embodiments, the equilibration buffer contains at least 10 to 100 mM Na. + or K + (For example, between 10-150 mM, 20-200 mM, or 100-300 mM). In some embodiments, the equilibration buffer is 20 mM HEPES-KOH (pH 7.3). In some embodiments, the equilibration buffer is phosphate-buffered saline (PBS = 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.8).

[0036] The mixture may also contain one or more surfactants. Sufficient surfactant may be included to stabilize the couplings and prevent aggregation and precipitation of the couplings in the mixture, especially when a high ionic strength buffer is introduced, which could otherwise lead to aggregation or precipitation of the couplings. In some embodiments, the surfactant is a nonionic polyalkylene glycol surfactant, such as polyethylene glycol. In some embodiments, the surfactant is a surfactant containing polyoxypropylene, such as a poloxamer surfactant. Poloxamer surfactants are characterized by a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyethylene oxide (poly(ethylene oxide)). Because the length of the polymer block can be customized, many different poloxamers exist, each with slightly different properties. Poloxamer copolymers are typically named with the letter "P" (representing poloxamer) followed by three digits. The first two digits multiplied by 100 give the approximate molecular weight of the polyoxypropylene core, and the last digit multiplied by 10 gives the percentage of polyoxyethylene content (e.g., P407 = poloxamer with a polyoxypropylene molecular weight of 4,000 g / mol and 70% polyoxyethylene content). For the trade names Pluronic and Synperonic poloxamers, these copolymers are coded with a single letter to define their physical form at room temperature (L = liquid, P = paste, F = flake (solid)), followed by two or three digits. The first digit in the numerical name (two of the three digits) multiplied by 300 represents the approximate molecular weight of the hydrophobic chain; the last digit multiplied by 10 gives the percentage of polyoxyethylene content (e.g., F-68 represents a polyoxypropylene molecular weight of 1,800 g / mol and 80% polyoxyethylene content). Exemplary poloxamer surfactants include, but are not limited to, Pluronic. F-68. The concentration of the surfactant used can be determined empirically (i.e., titration to prevent precipitation of the coupling compound). In some embodiments, the surfactant concentration is 0.02%-1%, for example, 0.05-0.2%, for example, 0.1%.

[0037] Before contacting a sample (e.g., a mixture of coupling compounds) with a polymer-filled chromatographic resin, the resin can be equilibrated to establish suitable pH, conductivity, and / or salt concentration.

[0038] After the sample is contacted with a polymer-filled chromatographic resin, target molecules (e.g., protein-nanoparticle conjugates) are expelled from the polymer-filled resin pores and collected in the resin flowthrough. Contaminants, such as free antibodies (and optionally uncoupled nanoparticles), are captured by the resin groups in the bead pores.

[0039] The presence of target molecules or other components of the sample in the resin output can be monitored as needed to determine fractions containing the target molecules, which are free of contaminants or at least have reduced levels of contaminants compared to the original sample. In some embodiments, at least 90%, 95%, or 99% of the contaminants in the sample are removed in the resulting purified target molecule fraction. Exemplary methods for measuring the output include monitoring the characteristic absorption wavelength of the target molecule. The term "fraction" is used to refer to a portion of the chromatographic output and is not intended to limit the manner of collection of the output or whether the output is a partial or continuous collection.

[0040] Example

[0041] Example 1 - Preparation and characterization of 3% agarose-filled hydroxyapatite microspheres (HAM)

[0042] Preparation of agarose-filled HAM:

[0043] A 3% agarose solution was prepared by dispersing 4.0 g of Hispanagar D5 (high gel strength) in 140 mL of water at 100 °C. 210 g of dry HAM (Bio-RadXT type) and 700 mL of heated molten agarose solution were mixed in a 700 mL flask and stirred for 60 minutes to form an insoluble gel within the pores of the HAM. The heated solution was then atomized by passing it through a nozzle with an inner diameter of 2 mm surrounded by an airflow of approximately 20 L / min at approximately 25 °C. The resulting particles were then collected in a water bath at approximately 20 °C–30 °C. The particles were then sieved using 120 μm and 65 μm sieves to remove excess agarose flakes. The sieved agarose-filled HAM was then suspended in water to remove excess agarose. (Reference) Figure 1A (Image of beads using an optical microscope) The resulting agarose-filled HAM contains individual HAMs with agarose in the wells, but no HAM clusters within the agarose. To demonstrate the presence of agarose in the pores of the HAM, the agarose-filled HAM was treated with acetate buffer at pH 3.5 to disperse the hydroxyapatite, and then stained. See also Figure 1B Image of a bead obtained using an optical microscope.

[0044] Characterization of agarose-filled HAM:

[0045] Assay for static binding capacity of thyroglobulin

[0046] Resuspend the entire final resin product in water. Transfer some slurry to a centrifuge column until the resin bed reaches the 0.5 ml mark (draw liquid from the bottom of the column with a syringe and tap the side of the column until the medium settles). Dry the resin under vacuum for 20 seconds. Then add 1 ml of ultrapure water, mix thoroughly with the resin, and transfer 0.3 ml of slurry to a new column. Wash the resin in the new column under vacuum with 5 ml of 10 mM MES (pH 6.5). Dry the resin under vacuum for 20 seconds. Transfer the semi-dry resin to a 2-ml Eppendorf tube. Add 1 ml of thyroglobulin sample to the tube. Place the tube on a rotor mixer and rotate for 60 minutes. Then centrifuge the tube at 3000 rpm for 4 minutes. Measure the UV absorbance of the supernatant and thyroglobulin sample at 280 nm using a spectrophotometer. Calculate the static binding capacity (SBC) using the following equation:

[0047] SBC (mg / mL) = (Total protein used (mg) - Unbound protein (mg)) / Resin volume (mL)

[0048] UV absorbance is used to determine protein concentration, and the molecular weight of the protein is used to determine the protein in mg.

[0049] The SBC results are shown in Table 1.

[0050] Determination of static binding capacity of γ-globulin

[0051] Resuspend the entire final resin product in water. Transfer some slurry to a centrifuge column until the resin bed reaches the 0.5 ml mark (draw liquid from the bottom of the column with a syringe and tap the side of the column until the medium settles). Dry the resin under vacuum for 20 seconds. Then add 1 ml of ultrapure water, mix thoroughly with the resin, and transfer 0.3 ml of slurry to a new column. Wash the resin in the new column under vacuum with 5 mL of 20 mM Tris-HCl and 150 mM NaCl (pH 7.5). Dry the resin under vacuum for 20 seconds. Transfer the semi-dry resin to a 2-ml Eppendorf tube. Add 1 ml of γ-globulin sample to the tube. Place the tube on a rotary mixer and rotate for 60 minutes. Centrifuge the tube at 3000 rpm for 4 minutes. Measure the UV absorbance of the supernatant and γ-globulin sample at 280 nm using a spectrophotometer. Static binding capacity (SBC) results are shown in Table 1.

[0052] Table 1

[0053]

[0054] Referring to Table 1, for agarose-filled resins, agarose prevents proteins from entering the pores of the microspheres to bind to the inner surface. Therefore, the SBC is reduced compared to the corresponding control samples. The larger the protein size, the greater the reduction in SBC. Thyroglobulin and gamma globulin have molecular weights of 660 kDa and 150 kDa, respectively. Therefore, for agarose-filled resins, the larger thyroglobulin shows a greater reduction in SBC than gamma globulin. The slight reduction in SBC for gamma globulin indicates that the smaller-sized protein, gamma globulin, can enter the pores to bind to the resin.

[0055] Example 2: Comparison of chromatographic characteristics between agarose-filled HAM and HAM

[0056] The purpose of this embodiment is to determine whether the chromatographic properties of HAM are altered by filling the pores of the HAM with agarose. An IgG-pdot conjugate (with a diameter of approximately 25 nm) was applied to each resin to determine whether the conjugate bound to the resin or excluded from the pore volume. Uncoupled pdot was also applied to 3% agarose-filled HAM to determine whether the agarose-filled pores allowed uncoupled pdot to enter the pores of the agarose-filled HAM and be captured by the agarose-filled HAM.

[0057] Comparison of chromatographic characteristics

[0058] Four disposable columns (Bio-Rad Micro Bio-Spin) TM The chromatographic column (used in gravity mode) was packed with 0.5 mL of resin as described in Table 1. The column was equilibrated with 1–2 mL of HEPES buffer (pH 7.3) containing 0.1% pluronic F68 (Thermo Fisher Scientific). 500 μL of purified goat anti-rabbit IgG-pdot conjugate sample or unconjugated pdot sample (in HEPES buffer) was applied to each column. The conjugate was prepared by modifying the pdot with maleimide, followed by linking the pdot to IgG via a thiol group (generated by treating the antibody with Tarwich's reagent). The pdot absorbed at 470 nm and had a reddish-brown color; therefore, the conjugate was reddish-brown.

[0059] After applying the sample, wash the column with 2 mL of HEPES buffer. Sample binding to the resin was determined visually; a red tinge at the top of the column or a red tinge dispersed throughout the resin indicated sample binding. If the column remained white, the sample had not bound to the resin (e.g., the sample was not bound). The results are summarized in Table 2.

[0060] Table 2

[0061]

[0062] The results in Table 2 indicate that the IgG-pdot conjugate binds to the HAM but not to the 3% agarose-filled HAM. Therefore, filling the wells with 3% agarose and coating the HAM surface prevents large conjugates from binding to the HAM while allowing smaller unconjugated pdots to enter the wells and bind to the surface within the HAM wells.

[0063] Example 3 - Preparation and characterization of 3% agarose-filled UNOsphere Q

[0064] Preparation of agarose-UNOsphere Q

[0065] In a 250 ml flask, a 3% agarose solution was prepared by dispersing 3.0 g of Hispanagar D5 (high gel strength) in 100 mL of water at 100 °C. 15 mL of UNOsphere (UNOsphere™ Q strong anion exchange medium from Bioradiation Laboratory Inc.) was added to the 250 ml flask and stirred for 60 minutes to form an insoluble gel within the medium pores. The heated solution was then atomized by passing it through a nozzle with an inner diameter of 2 mm surrounded by an airflow of approximately 20 L / min at approximately 25 °C. The resulting particles were then collected in a water bath at approximately 20 °C–30 °C. The particles were then sieved using a 120 μm sieve to remove excess agarose flakes. The sieved agarose-filled UNOsphere was then suspended in water to remove excess agarose. The resulting agarose-filled UNOsphere contained separate UNOsphere beads with agarose in the pores, without clusters of UNOsphere beads within the agarose (see [link to product description]). Figure 2 (Image of UNOsphere beads filled with agarose obtained by optical microscopy).

[0066] Characterization of agarose-UNOsphere Q

[0067] Assay for static binding capacity of thyroglobulin

[0068] Resuspend the entire final resin product in water. Transfer some slurry to a centrifuge column until the resin bed reaches the 0.5 ml mark (draw liquid from the bottom of the column with a syringe and tap the side of the column until the medium settles). Dry the resin under vacuum for 20 seconds. Then add 1 ml of ultrapure water, mix thoroughly with the resin, and transfer 0.3 ml of slurry to a new column. Wash the resin in the new column under vacuum with 5 ml of 20 mM Bis-Tris and 50 mM NaCl (pH 7.0). Dry the resin under vacuum for 20 seconds. Transfer the semi-dry resin to a 2-ml Eppendorf tube, and then add 1 ml of thyroglobulin sample to the tube. Place the tube on a rotor mixer and rotate for 30 minutes. Centrifuge the tube at 3000 rpm for 4 minutes. Measure the UV absorbance of the supernatant and thyroglobulin sample at 280 nm using a spectrophotometer. Static binding capacity (SBC) results are shown in Table 2.

[0069] Determination of static binding capacity of γ-globulin

[0070] Resuspend the entire final resin product in water. Transfer some slurry to a centrifuge column until the resin bed reaches the 0.5 ml mark (draw liquid from the bottom of the column with a syringe and tap the side of the column until the medium settles). Dry the resin under vacuum for 20 seconds. Then add 1 ml of ultrapure water, mix thoroughly with the resin, and transfer 0.3 ml of slurry to a new column. Wash the resin in the new column under vacuum with 5 mL of 20 mM Tris-HCl (pH 9.5). Dry the resin under vacuum for 20 seconds. Transfer the semi-dry resin to a 2-ml Eppendorf tube. Add 1 ml of γ-globulin sample to the tube. Place the tube on a rotary mixer and rotate for 30 minutes. Centrifuge the tube at 3000 rpm for 4 minutes. Measure the UV absorbance of the supernatant and γ-globulin sample at 280 nm using a spectrophotometer. Calculate the SBC as described above. The SBC results are shown in Table 3.

[0071] Table 3

[0072]

[0073] The results for agarose-filled UNOsphere Q in Table 3 show that larger thyroglobulins resulted in a greater decrease in SBC compared to gamma globulins. This indicates that larger proteins are prevented from entering the pores of the agarose-filled resin to bind to the inner surface.

[0074] Example 4: Comparison of chromatographic characteristics between agarose-filled UNOsphere Q and UNOsphere Q

[0075] The purpose of this embodiment is to determine whether the chromatographic properties of UNOsphere Q are altered by passing the pores of UNOsphere Q through agarose filling. An IgG-pdot conjugate (with a diameter of approximately 25 nm) was applied to each resin to determine whether the conjugate bound to the resin or excluded from the pore volume. Unconjugated pdot was also applied to 3% agarose-filled UNOsphere Q to determine whether the agarose-filled pores allowed unconjugated pdot to enter and be captured by the agarose-filled UNOsphere Q.

[0076] Comparison of chromatographic performance

[0077] Four disposable columns (Bio-Rad Micro Bio-Spin) TM The chromatographic column (used in gravity mode) was packed with 0.25 mL of resin as described in Table 1. The column was equilibrated with 1–2 mL of 20 mM Tris-HCl buffer (pH 8.5). 500 μL of purified goat anti-rabbit IgG-pdot conjugate sample or unconjugated pdot sample (in 20 mM Tris-HCl buffer) was applied to each column. The conjugate was prepared by modifying pdot with maleimide and then linking pdot to IgG via a thiol group (generated by treating the antibody with Tarwich's reagent). The pdot absorbed at 470 nm and had a reddish-brown color; therefore, the conjugate was reddish-brown.

[0078] After applying the sample, wash the column with 1 mL of Tris-HCl buffer. Sample binding to the resin was determined visually; a red tinge at the top of the column or a red tinge dispersed throughout the resin indicated sample binding. If the column remained white, the sample had not bound to the resin (e.g., no sample binding). The results are summarized in Table 4.

[0079] Table 4

[0080]

[0081] The results in Table 4 indicate that the IgG-pdot conjugate and Pdot bind to UNOsphere Q, but not to 3% agarose-filled UNOsphere Q. Therefore, filling the pores of UNOsphere Q with 3% agarose and coating its surface prevents the conjugate-Pdot and Pdot from binding to UNOsphere Q.

[0082] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art will understand that various modifications or changes can be made accordingly, and these are included within the scope of the spirit and benefits of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for preparing a polymer-filled chromatographic resin, wherein the chromatographic resin is hydroxyapatite microspheres, the hydroxyapatite microspheres are XT-type hydroxyapatite microspheres, and the method comprises: The chromatographic resin is added to a solution containing molten polymer while stirring to allow the heated polymer to absorb or otherwise enter the pores of the chromatographic resin. The solution is atomized, and insoluble porous polymer-filled chromatographic resin beads are collected in a water bath. and Excess polymer is removed from the insoluble porous polymer-filled chromatographic resin beads by passing them through one or more sieves with openings of 50 µm to 200 µm.

2. The method of claim 1, further comprising washing the insoluble porous polymer-filled chromatographic resin beads.

3. The method according to any one of claims 1-2, wherein the polymer is agarose and the concentration of the agarose is 0.5% to 8%.

4. The method according to any one of claims 1-2, wherein the chromatographic resin comprises acrylamide, methacrylate, polystyrene, or silica.

5. The method of claim 4, wherein the chromatographic resin comprises a copolymer of 3-allyloxy-1,2-propanediol and vinylpyrrolidone crosslinked with N,N'-methylenebisacrylamide.

6. The method of claim 4, wherein the chromatographic resin comprises glycidyl methacrylate and diethylene glycol dimethacrylate.

7. The method of claim 1, wherein the molten polymer is at at least 100°C.

8. The method of claim 1, wherein atomizing the solution comprises surrounding the solution ejected from the nozzle with an airflow.

9. The method of claim 8, wherein the temperature of the airflow is from 20°C to 90°C.

10. The method of claim 8 or 9, wherein the airflow velocity is from 15 L / min to 25 L / min.

11. The method of claim 1, wherein the temperature of the water bath is from 4°C to 40°C.

12. Use of the polymer-filled chromatographic resin prepared by the method of any one of claims 1-11 for chromatographic analysis, comprising: A sample containing the target molecule is contacted with a polymer-filled chromatographic resin, the contact being performed under conditions that prevent the target from being trapped by the insoluble porous polymer-filled chromatographic resin; and The target molecules are collected from the insoluble porous polymer-filled chromatographic resin.

13. The use as claimed in claim 12, wherein the sample contains contaminants captured by a chromatographic resin filled with an insoluble porous polymer.

14. The use as described in claim 13, wherein the target molecule is a protein-nanoparticle conjugate and the contaminant is a free protein.

15. The use as claimed in claim 12, wherein the sample comprises a protein-nanoparticle conjugate, free protein, and buffer solution.

16. The use as claimed in claim 15, wherein the sample further comprises a surfactant.

17. The use as described in claim 16, wherein the surfactant is a polyalkylene glycol.

18. The use as described in claim 17, wherein the surfactant is a poloxamer surfactant.

19. The use as described in claim 12, wherein the target molecule is a virus.

20. The use as claimed in claim 12, wherein the collection step comprises collecting one or more fractions rich in the target molecule from a chromatographic resin filled with the insoluble porous polymer.

21. The use as claimed in claim 12, wherein the collection step comprises applying centrifugation or vacuum to the chromatographic resin filled with the insoluble porous polymer and collecting one or more fractions rich in the target molecule from the chromatographic resin filled with the insoluble porous polymer.

22. The use as described in any one of claims 14-21, wherein the protein is an IgG antibody.

23. The use as described in any one of claims 14-21, wherein the protein-nanoparticle conjugate is a protein-polymer dot conjugate.

24. The use according to any one of claims 14-21, wherein the sample further comprises free nanoparticles, and the insoluble porous polymer-filled chromatographic resin separates the free nanoparticles from the conjugate.

25. The use as described in any one of claims 12-21, wherein the chromatographic resin is ceramic hydroxyapatite.

26. The use according to any one of claims 12-21, wherein the chromatographic resin is a macroporous resin comprising a copolymer of 3-allyloxy-1,2-propanediol and vinylpyrrolidone crosslinked with N,N'-methylenebisacrylamide.

27. The use according to any one of claims 12-21, wherein the polymer is agarose at a concentration of 0.5% to 8%.

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