Protein A / G particles for affinity chromatography and method of using the same

Abstract

This disclosure relates to non-porous polymer particles having an average particle size of 1 to 10 micrometers and functionalized with immunoglobulin-binding proteins. The functionalized particles of this disclosure can be used to purify immunoglobulins within a range of affinity for immunoglobulin-binding proteins.

Description

[Technical Field]

[0001] This application claims the interests of U.S. Provisional Application No. 63 / 464,384, filed on 5 May 2023, and U.S. Provisional Application No. 63 / 587,864, filed on 4 October 2023, both of which are incorporated herein by reference in their entirety.

[0002] This technology relates to the capture of substances by affinity. More specifically, this technology relates to chromatography columns using non-porous particles used in affinity chromatography. [Background technology]

[0003] Chromatography is a separation technique with broad applicability across various industries, including pharmaceuticals, biotechnology, and the chemical industry. Chromatography requires two phases: a stationary phase and a mobile phase. Typically, the stationary phase contains porous or non-porous particles packed into a column. The mobile phase then carries the sample through the stationary phase column. Affinity chromatography, a specific application of chromatography, utilizes functionalized particles in the stationary phase. The fundamental principle of affinity chromatography is that compounds or specimens in a sample have distinct affinities to functional groups on the particles. These differences allow for the separation, isolation, and concentration of specific specimens from complex heterogeneous samples.

[0004] As an example, the target sample isolated using affinity chromatography is an antibody. Antibodies are typically isolated from culture media (e.g., hybridoma cultures), serum, or ascites fluid, each containing samples of unpredictable numbers and compositions. However, due to the complexity and / or volume of the antibody-containing sample, the antibody may be at a concentration too low to be detected using standard analytical methods, or in the presence of significant impurities. Furthermore, the practical applications of antibodies in experimental and therapeutic applications often require homogeneous, pure, and concentrated samples. Therefore, it is highly desirable to be able to isolate antibodies that are substantially pure and at a concentration high enough to be used downstream in any applicable assay.

[0005] Current affinity chromatography methods suffer from low throughput and high costs due to columns, often resulting in antibody loss due to nonspecific binding and / or insufficient antibody retention. Other types of immunoassays, such as ELISA, are complex, time-consuming, and highly variable. Therefore, there is a need in this field for affinity chromatography columns that are highly efficient and enable robust separation, collection, and concentration of various antibodies regardless of their target antigens. [Overview of the project] [Problems that the invention aims to solve]

[0006] (Summary of the invention) Generally, this technology relates to a plurality of particles used in affinity chromatography. More specifically, this technology relates to a chromatography column containing a plurality of particles having an average particle size of less than 10 μm, wherein the hydrophilic outer surface of each particle is conjugated with an immunoglobulin-binding protein, such as protein A, which functions as a binding site for an antibody. As a result, the chromatography column of this technology can be used for antibody affinity chromatography. [Means for solving the problem]

[0007] In one embodiment, the present technology relates to a chromatography column. The chromatography column comprises a column body formed of a metal or metal alloy and accommodates a plurality of particles. Each of the plurality of particles comprises a nonporous polymer core, a hydrophilic surface on the outer layer of the nonporous polymer core, and one or more molecules of immunoglobulin-binding domains conjugated to the hydrophilic surface, and the particles have an average particle size of 1.5 μm to 8 μm.

[0008] Other embodiments of this technology are as follows: In some embodiments, the immunoglobulin-binding protein is protein A, protein G, protein A / G, or protein L, or their binding domains. In some embodiments, the non-porous polymer core has a gradient composition. In some embodiments, the non-porous polymer core contains divinylbenzene (80%). In some embodiments, the hydrophilic surface is (3-glycidyloxypropyl)trimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, polyacrylate, glycidol, glycerol triglycidyl ether, butyl diglycidol ether, or poly(methyl acrylate). In certain embodiments, one or more molecules of the immunoglobulin-binding protein are conjugated to the hydrophilic surface of the particles via an epoxy linker. In some embodiments, the average particle size is 2 to 5 μm. In one embodiment, the average particle size is 3.5 μm. In some embodiments, the immunoglobulin-binding protein has a surface coverage of 3 to 9 μg per mg of particles. In some embodiments, the immunoglobulin-binding protein is protein A, and has a surface coverage of 3 to 9 μg per mg of particles.

[0009] In some embodiments, the chromatography column is coated with an alkylsilyl material on at least a portion of the inner surface of the column body. In some embodiments, the column body includes a frit, which is coated with an alkylsilyl material. In some embodiments, the alkylsilyl material is a hydrophilic, nonionic layer of polyethylene glycol silane.

[0010] In one embodiment, a chromatography device is disclosed herein that includes a chromatography column, a column injector located upstream of the chromatography column, and piping fluidly connected to the chromatography column and located downstream thereof, wherein a portion of the inner surface of the column injector and a portion of the inner surface of the piping are coated with an alkylsilyl material.

[0011] In one embodiment, a method for enriching with immunoglobulin is disclosed herein, the method comprising four steps a) to d). Step a) comprises selecting a chromatography column, the chromatography column comprising a column body formed of metal or a metal alloy, the column body containing a plurality of particles, each particle comprising a non-porous polymer core, a hydrophilic surface on the outer layer of the non-porous polymer core, and one or more molecules of immunoglobulin-binding protein conjugated on the hydrophilic surface, the particles having an average particle size of 1.5 to 8 μm. Step b) comprises washing the chromatography column with a binding buffer. Step c) comprises applying a solution containing immunoglobulin to the chromatography column. Step d) comprises washing the chromatography column with an elution buffer so that the immunoglobulin is eluted from the chromatography column.

[0012] In some embodiments, the immunoglobulin-binding protein is protein A, protein G, protein A / G, protein L, or their binding domains. In some embodiments, the chromatography column is connected to a high-performance liquid chromatography (HPLC) system, an ultra-high-performance liquid chromatography (UHPLC) system, or a high-performance protein liquid chromatography (FPLC) system. In some embodiments, the elution buffer is at least three orders of magnitude (1000 times) more acidic than the binding buffer. In some embodiments, the binding buffer has a pH of 7.0–8.0. In some embodiments, the elution buffer has a pH of 1.3–3.5.

[0013] In some embodiments, the method further includes step e) detecting immunoglobulins with an ultraviolet spectrometer, a fluorescence spectrometer, and / or a mass spectrometry detector. In some embodiments, the method further includes step f) washing a chromatography column with a binding buffer. In some embodiments, the method further includes repeating steps b) to e).

[0014] In other embodiments, the method further includes step e) washing the chromatography column with binding buffer. In some embodiments, the method further includes repeating steps b) to d).

[0015] In some embodiments, the chromatography column is washed with binding buffer for 10 minutes or less. In some embodiments, the method can be carried out without requiring organic modifiers to reduce nonspecific binding.

[0016] In some embodiments, the step of applying the immunoglobulin-containing solution to the chromatography column (step c) is performed for about 1 to 3 minutes. In some embodiments, the step of applying the immunoglobulin-containing solution to the chromatography column (step c) is performed for about 1 minute.

[0017] This technology will be more fully understood from the following detailed description in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0018] [Figure 1A] Figure 1A is a cross-sectional view of particles prior to the attachment of an immunoglobulin-binding protein according to one embodiment of the present technology. [Figure 1B] Figure 1B is a cross-sectional view of the particles of Figure 1A after the attachment of an immunoglobulin-binding protein according to one embodiment of the present technology. [Figure 2A] Figure 2A is a perspective view of a chromatography column filled with a plurality of particles (i.e., the particles of Figure 1B) having a notch (220) for illustrating the interior of the chromatography column. [Figure 2B] Figure 2B is a cross-sectional view of the chromatography column of Figure 2A taken along line BB. [Figure 3] Figure 3 is a schematic diagram explaining a method of capturing an antibody by affinity using the chromatography column of Figure 2B. [Figure 4A] Figure 4A shows a comparison of the peak areas (black solid line) of IgG eluted at various concentrations from 0.25 to 20 μg for the column of Figure 2 with a comparator column 1 column (dotted line). [Figure 4B] Figure 4B shows a comparison of the peak areas (black solid line) of IgG eluted at various concentrations from 0.25 to 300 μg for the column of Figure 2 with a comparator column 1 column (dotted line). [Figure 4C] Figure 4C shows the percent carryover of IgG between affinity capture assays compared to comparator column 1 for the column of Figure 2. [Figure 5A] Figure 5A shows a comparison of the elution peak (black dashed line) of IgG eluted for the column of Figure 2 with a comparator column 3 column (dashed line) and a comparator column 2 column (solid line). [Figure 5B]Figure 5B shows the percent carry-over of IgG between affinity capture assays compared to comparison column 2 and comparison column 3 for the column of Figure 2. [Figure 6A] Figure 6A shows the peak area of IgG eluted using a 1-minute binding step (black solid line) or a 3-minute binding step (black dashed line) for the column of Figure 2. [Figure 6B] Figure 6B shows the quantified peak area of IgG eluted using a 1-minute binding step or a 3-minute binding step over various concentrations of IgG for the column of Figure 2. [Figure 6C] Figure 6C shows the percent carry-over of IgG between affinity capture assays using a 1-minute binding step or a 3-minute binding step over various concentrations of IgG for the column of Figure 2. [Figure 7A] Figures 7A - 7C show the chromatograms of monoclonal antibodies eluted from the column of particle type B using a phosphate elution method (Figure 7A) or a glycine elution method (Figure 7B). Figure 7C shows the linear correlation between the monoclonal antibody concentration and the peak area. [Figure 7B] Figures 7A - 7C show the chromatograms of monoclonal antibodies eluted from the column of particle type B using a phosphate elution method (Figure 7A) or a glycine elution method (Figure 7B). Figure 7C shows the linear correlation between the monoclonal antibody concentration and the peak area. [Figure 7C] Figures 7A - 7C show the chromatograms of monoclonal antibodies eluted from the column of particle type B using a phosphate elution method (Figure 7A) or a glycine elution method (Figure 7B). Figure 7C shows the linear correlation between the monoclonal antibody concentration and the peak area. [Figure 8A] Figures 8A - 8C show the chromatograms of monoclonal antibodies eluted from the column of particle type D using a phosphate elution method (Figure 8A) or a glycine elution method (Figure 8B). Figure 8C shows the linear correlation between the monoclonal antibody concentration and the peak area. [Figure 8B]Figures 8A-8C show chromatographs of monoclonal antibodies eluted from particle type D columns using either a phosphate elution method (Figure 8A) or a glycine elution method (Figure 8B). Figure 8C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 8C] Figures 8A-8C show chromatographs of monoclonal antibodies eluted from particle type D columns using either a phosphate elution method (Figure 8A) or a glycine elution method (Figure 8B). Figure 8C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 9A] Figures 9A-9C show chromatographs of monoclonal antibodies eluted from particle type H columns using either a phosphate elution method (Figure 9A) or a glycine elution method (Figure 9B). Figure 9C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 9B] Figures 9A-9C show chromatographs of monoclonal antibodies eluted from particle type H columns using either a phosphate elution method (Figure 9A) or a glycine elution method (Figure 9B). Figure 9C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 9C] Figures 9A-9C show chromatographs of monoclonal antibodies eluted from particle type H columns using either a phosphate elution method (Figure 9A) or a glycine elution method (Figure 9B). Figure 9C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 10A] Figures 10A-10C show chromatographs of monoclonal antibodies eluted from particle type E columns using either a phosphate elution method (Figure 10A) or a glycine elution method (Figure 10B). Figure 10C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 10B] Figures 10A-10C show chromatographs of monoclonal antibodies eluted from particle type E columns using either a phosphate elution method (Figure 10A) or a glycine elution method (Figure 10B). Figure 10C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 10C]Figures 10A-10C show chromatographs of monoclonal antibodies eluted from particle type E columns using either a phosphate elution method (Figure 10A) or a glycine elution method (Figure 10B). Figure 10C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 11A] Figures 11A-11C show chromatographs of monoclonal antibodies eluted from particle type C columns using either a phosphate elution method (Figure 11A) or a glycine elution method (Figure 11B). Figure 11C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 11B] Figures 11A-11C show chromatographs of monoclonal antibodies eluted from particle type C columns using either a phosphate elution method (Figure 11A) or a glycine elution method (Figure 11B). Figure 11C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 11C] Figures 11A-11C show chromatographs of monoclonal antibodies eluted from particle type C columns using either a phosphate elution method (Figure 11A) or a glycine elution method (Figure 11B). Figure 11C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 12A] Figures 12A-12C show chromatographs of monoclonal antibodies eluted from particle type F columns using either a phosphate elution method (Figure 12A) or a glycine elution method (Figure 12B). Figure 12C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 12B] Figures 12A-12C show chromatographs of monoclonal antibodies eluted from particle type F columns using either a phosphate elution method (Figure 12A) or a glycine elution method (Figure 12B). Figure 12C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 12C] Figures 12A-12C show chromatographs of monoclonal antibodies eluted from particle type F columns using either a phosphate elution method (Figure 12A) or a glycine elution method (Figure 12B). Figure 12C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 13A]Figures 13A-13C show chromatographs of monoclonal antibodies eluted from particle type A columns using either a phosphate elution method (Figure 13A) or a glycine elution method (Figure 13B). Figure 13C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 13B] Figures 13A-13C show chromatographs of monoclonal antibodies eluted from particle type A columns using either a phosphate elution method (Figure 13A) or a glycine elution method (Figure 13B). Figure 13C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 13C] Figures 13A-13C show chromatographs of monoclonal antibodies eluted from particle type A columns using either a phosphate elution method (Figure 13A) or a glycine elution method (Figure 13B). Figure 13C shows the linear correlation between monoclonal antibody concentration and peak area. [Figure 14A-1] Figures 14A to 14B show chromatographs comparing the peak shapes for particle types A, B, C, D, E, F, and H using either the phosphoric acid elution method (Figure 14A) or the glycine elution method (Figure 14B). [Figure 14A-2] Figures 14A to 14B show chromatographs comparing the peak shapes for particle types A, B, C, D, E, F, and H using either the phosphoric acid elution method (Figure 14A) or the glycine elution method (Figure 14B). [Figure 14B-1] Figures 14A to 14B show chromatographs comparing the peak shapes for particle types A, B, C, D, E, F, and H using either the phosphoric acid elution method (Figure 14A) or the glycine elution method (Figure 14B). [Figure 14B-2] Figures 14A to 14B show chromatographs comparing the peak shapes for particle types A, B, C, D, E, F, and H using either the phosphoric acid elution method (Figure 14A) or the glycine elution method (Figure 14B). [Figure 15A] Figures 15A and 15B compare the peak heights (Figure 15A) and peak volumes (Figure 15B) for particle types A, B, C, D, E, F, and G for both elution methods. [Figure 15B]Figures 15A and 15B compare the peak heights (Figure 15A) and peak volumes (Figure 15B) for particle types A, B, C, D, E, F, and G for both elution methods. [Modes for carrying out the invention]

[0019] To make this technology easier to understand, several terms are defined first. In addition, whenever a value or range of a parameter is given, it should be noted that intermediate values ​​and ranges up to the given value are also intended to be part of this disclosure. The word “about” means ±5% unless otherwise specified. It should also be noted that, as used herein and in the claims, the singular forms “a,” “an,” and “the” also encompass plural references unless otherwise clearly indicated.

[0020] definition As used herein, the terms “nonporous” or “nonporous core” refer to a material or a portion of a material (e.g., a core) having a pore volume of less than 0.1 cc / g. Preferably, in some embodiments, a nonporous polymer core has a pore volume of less than 0.10 cc / g (e.g., 0.05 cc / g), preferably less than 0.02 cc / g. The pore volume is determined using methods known in the art based on multipoint nitrogen adsorption experiments (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, GA).

[0021] As used herein, the term “hard particles” refers to the strength of particles in withstanding pressures applied under flow conditions. Hard particles appear visually undamaged in scanning electron microscope images after exposure to a pressure of 3,500 psi, at which less than 10% of observed particles are visually damaged (i.e., they maintain the same shape factor without being broken, crushed, or altered). In addition, broken or deformed particles in a packed bed cause a decrease in flow and an increase in pressure, as predicted using the Kozeny-Carmen formula. Broken or deformed particles in a packed bed can increase pressure beyond levels suitable for use in HPLC or UHPLC.

[0022] As used herein, the term "conjugated" refers to a bond between two molecules formed by a chemical bond between a reactive functional group of one molecule, such as protein A, and a moderately reactive functional group of another molecule, such as an epoxide.

[0023] As used herein, the term "conjugate" refers to a compound formed by the chemical bonding of a reactive functional group of one molecule, such as protein A, with a moderately reactive functional group of another molecule, such as an epoxide. An example of a moderately reactive functional group is a nucleophile / electrophile pair. For example, the nucleophile may be an amine or thiol group derived from an amino acid of protein A, and the electrophile may be an epoxide.

[0024] As used herein, the terms “immunoglobulin” and “antibody” are used synonymously unless otherwise specified.

[0025] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive to a particular antigen. This includes polyclonal, monoclonal, genetically modified, and other denatured forms of antibodies, including, but not limited to, chimeric antibodies, camelid antibodies, monobodies, humanized antibodies, and heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, bispecific antibodies). Unless otherwise indicated, the term “monoclonal antibody” includes both the intact molecule and the antibody fragment that can specifically bind to a target protein.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as they would be generally understood by those with ordinary skill in the art to which this disclosure belongs.

[0027] material This technology relates to materials, columns, and devices used in affinity chromatography. In particular, this technology relates to materials used in high-performance liquid chromatography (HPLC) systems or ultra-high-performance liquid chromatography (UHPLC) systems, and is adapted to enable affinity-based capture under the high-pressure and flowing conditions associated with HPLC and / or UHPLC.

[0028] In this technology, multiple particles are contained within a chromatography column. Along with other components, these particles are designed to provide on-column affinity capture under the high pressure and flow rates associated with HPLC and UHPLC systems.

[0029] particle To provide a surface area for stability and affinity-based capture, the particles of this technology are nonporous. Nonporous particles provide a suitable surface area for adhesion or coating by one or more affinity-binding groups. In some embodiments, each particle in a plurality of particles packed into a column may be highly spherical and have a smooth surface. In some embodiments, each particle in a plurality of particles packed into a column may be highly spherical and have a rough, convex surface. Such materials have a surface area (m²) close to their theoretical value. 2 It has (measured in / g). The theoretical surface area for a non-porous smooth sphere is equal to 6 / {particle diameter × particle density}. For example, a 1-micron polymer particle with a density of approximately 1 g / mL has a surface area of ​​6 m 2 A 3.5 micron polymer particle with the same density and a theoretical surface area of ​​1.7 m² / g is equivalent to 1.7 m². 2 A polymer particle with a theoretical surface area of ​​0.9 m² / g and the same density is 7 micrometers in size. 2 It has a theoretical surface area of ​​ / g.

[0030] While we do not wish to be bound by theory, the use of non-porous spheres is considered advantageous because it improves the dynamics of bonding and elution of affinity groups attached to the sphere's surface (whether smooth or with a convex or uneven surface). The shape factor of the non-porous sphere is thought to shut down the diffusion dynamics of particles into the pores.

[0031] The particles are nonporous. While some pores or porosity may be incorporated within the particles as discontinuous or microporous structures, nonporous particles are those having a pore volume of less than 0.1 cc per gram of the material forming the particle. Preferably, nonporous particles have a pore volume of less than 0.10 cc / g (e.g., 0.05 cc / g), preferably less than 0.02 cc / g in some embodiments. The pore volume is determined based on multipoint nitrogen adsorption experiments using methods known in the art (Micromeritics ASAP 2400; Micromeritics Instruments Inc., Norcross, GA).

[0032] The particles of this technology have an average particle size of less than 10 micrometers. For example, in one embodiment of this technology, the average particle size of the multiple particles packed into the column may be any value between 8 micrometers and 1.5 micrometers. In one embodiment, the average particle size of the multiple particles is 7 micrometers. In another embodiment, the average particle size is 3.5 micrometers. And in yet another embodiment, the average particle size is 1.7 micrometers.

[0033] Size (i.e., less than 10 micrometers), shape (i.e., spherical), and surface area (i.e., non-porous, smooth, or uneven convex outer surface) create shape factors useful for affinity-based capture from flowing samples. To achieve high throughput and minimize assay development, the particles of this technology are used with LC systems such as HPLC and UHPLC systems. These systems operate at high pressure (e.g., typically higher than 3,000 psi, such as 5,000 psi, 10,000 psi, 12,000 psi, 15,000 psi, and others). Consequently, the particles of this technology need to be rigid so that they retain their shape factors under HPLC and UHPLC operating conditions.

[0034] Generally, the particles produced by this technology are rigid particles that maintain their shape characteristics (e.g., undamaged, uncrushed, undestroyed, or unchanged) under HPLC or UHPLC operating conditions (e.g., pressure and flow rate). For example, the rigid particles produced by this technology do not visibly change in morphology (e.g., they do not break, crush, or change from their spherical shape), as can be confirmed using scanning electron microscopy before (i.e., control) and after the application of HPLC or UHPLC conditions.

[0035] Specific materials that meet shape factor considerations for forming the core (e.g., center or base) of the particles of this technology are polymers, particularly organic polymers. In some embodiments, the nonporous particles of this technology include a nonporous polymer core. In one embodiment, the nonporous polymer core of the particles is divinylbenzene (DVB), for example, 80% divinylbenzene. In some embodiments, the nonporous polymer core is formed to contain two or more polymers. For example, in some embodiments, the nonporous polymer core contains both divinylbenzene and polystyrene. In certain embodiments, the nonporous polymer core can be manufactured to include a gradient in polymer composition. For example, the inner portion of the core can be formed of 100% a first polymer (i.e., polymer A), and the outer portion of the core can be formed of a second polymer (i.e., polymer B) in a percentage somewhat greater than 100% or 0%. The percentages of polymer A and polymer B can change radially from the inner portion to the outer portion of the core to form a gradient in polymer composition. Other embodiments of nonporous polymer cores and particles suitable for use with this technology are described in U.S. Patent Application Publication No. 2019 / 0322783.

[0036] While the examples and embodiments of this technology illustrate the use of a non-porous polymer core for particles, it is known that other non-porous materials can be used, as long as the particle shape factor can be maintained under the operating conditions of HPLC or UPHLC. That is, other materials such as silica, metal oxides, hybrid inorganic-organic materials, or combinations thereof can be used to create non-porous spherical particles having an average particle size of less than 10 micrometers and possessing the rigidity or strength to maintain their shape factor under high operating pressure.

[0037] To form particles useful for affinity-based capture, the outer surface of the nonporous core of the particles is bonded or attached to affinity attachment groups. To do this, in one embodiment, the outer surface of the nonporous polymer core contains a hydrophilic material. That is, a hydrophilic surface is created in this outer region of the nonporous polymer core. One or more molecules of immunoglobulin-binding protein are conjugated to this hydrophilic surface. One or more molecules of immunoglobulin-binding protein provide an accessible binding site for immunoglobulins. In some embodiments, the immunoglobulin-binding protein is protein A, protein G, protein A / G, or protein L. In one embodiment, any immunoglobulin-binding protein that binds to a conserved portion of an antibody is suitable for use with this technology.

[0038] A hydrophilic surface can also be called a hydrophilic layer. The hydrophilic surface is located on the outer surface of a non-porous polymer core and can be formed from a polymer, molecule, or siloxane (e.g., hydroxyl, PEG, sugars, or carbohydrates) having a high density of hydrophilic groups. The immobilization of these hydrophilic groups can occur by condensation (esters, amides, silanols, silyl ethers), polymerization (methacrylates, acrylates, styryls), epoxy activation (epihydrochlorin), or ether formation (direct adhesion of PEG or carbohydrate groups by ether formation).

[0039] In one embodiment, the hydrophilic surface comprises a material selected from the group consisting of (3-glycidyloxypropyl)trimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, polyacrylate, poly(methylacrylate), and combinations thereof. In another embodiment, the hydrophilic surface comprises a material selected from the group consisting of glycidol, glycerol triglycidyl ether, and combinations thereof.

[0040] In embodiments in which immunoglobulin-binding proteins are used to provide a binding site for an antibody, linkers are typically used to immobilize or conjugate the immunoglobulin-binding protein to a hydrophilic surface. Such linkers include, but are not limited to, epoxy linkers, hydroxyl linkers, and any other linkers known in the art (see Hermanson G, "Bioconjugate Techniques," 3rd edition, July 2013).

[0041] Figure 1A shows an embodiment of a particle having a nonporous polymer core according to the present technology. That is, the particle shown in Figure 1A has shape factors (e.g., spherical, nonporous, and rigid) that can withstand the operating conditions of HPLC and UHPLC. The particle 100 shown in Figure 1A is a cross-sectional view prior to the addition of an immunoglobulin-binding protein, such as protein A. The particle 100 includes a nonporous polymer core 112 having an internal core region 105 and a radially extending region 110 surrounding the internal core region 105. The internal core region 105 is typically formed of one polymer or a uniform blend of several polymers, while the radially extending region 110 is typically formed of two or more polymers so as to form a gradient within this region. For example, the core region 105 can be formed of polystyrene, while the radially extending region 110 contains a gradient composition ranging from 100% polystyrene to 80% to 100% DVB with the remainder being polystyrene.

[0042] In one embodiment, the following three steps were used to form a non-porous polymer core 112. In step 1, 561.1 g of reagent alcohol (90% ethanol, about 5% methanol, and about 5% isopropanol), 16.9 g of polyvinylpyrrolidone (PVP-40, average molecular weight 40,000), 1.6 g of 2,2'-azobis(2-methylpropionitrile) (AIBN), 6.7 g of Triton® N-57, 80.1 g of styrene, and 2.4 g of poly(propylene glycol) dimethacrylate (average molecular weight 560) were charged into the reactor. After purging with nitrogen, the reaction mixture was heated to 70°C with stirring and maintained at 70°C until all reaction steps were completed. In Step 2, after the reaction mixture from Step 1 was kept at 70°C for 3 hours, a solution containing 52.0 g of DVB80, 24.0 g of styrene, 51.0 g of PVP-40, 1080.4 g of reagent alcohol (90% ethanol, approximately 5% methanol, and approximately 5% isopropanol), and 54.1 g of p-xylene was added to the reaction mixture at a constant flow rate over 2 hours. In Step 3, after the preparation of the solution from Step 2 was complete, a primer coating solution containing 31.2 g of glycidyl methacrylate (GMA), 6.2 g of ethylene glycol dimethacrylate (EDMA), 12.9 g of PVP-40, and 381.9 g of reagent alcohol (90% ethanol, approximately 5% methanol, and approximately 5% isopropanol) was added to the reaction mixture at a constant flow rate over 1.5 hours. After the reaction mixture was kept at 70°C for a total of 20 hours, the particles were separated from the reaction slurry by filtration. The particles were then washed with methanol, followed by tetrahydrofuran (THF), and then with acetone. The final product was dried overnight in a vacuum oven at 45°C. 91.8 g of monodisperse 2.3 μm polymer particles were obtained.

[0043] The embodiment shown in Figure 1A shows that the nonporous polymer core 112 has two regions (an internal core region 105 and a radially extending region 110), but this is not required. Other embodiments may feature a nonporous polymer core having only one region, i.e., the nonporous polymer core extends from the center of the particle to the outer surface of the nonporous polymer core 112.

[0044] As shown in Figure 1A, the hydrophilic surface or layer 115 is formed on the outer surface of the nonporous polymer core (i.e., opposite to the central region 105). In one embodiment, the hydrophilic surface 115 is formed by applying a hydrophilic primer coating solution containing 36.2 g of glycidyl methacrylate (GMA), 7.44 g of ethylene glycol dimethacrylate (EDMA), 8.21 g of PVP360 (PVP360, average molecular weight 360,000), and 489.4 g of reagent alcohol (90% ethanol, about 5% methanol, and about 5% isopropanol). This solution was added to a mixture containing the nonporous polymer core at a constant flow rate over about 1.5 hours to form the hydrophilic surface 115.

[0045] The above example is provided for illustrative purposes only. Other types of hydrophilic surfaces can be applied. For example, the hydrophilic layer may be formed of (3-glycidyloxypropyl)trimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, polyacrylate and / or poly(methylacrylate), glycidol, glycerol triglycidyl ether, butyl diglycidol ether, or any other type of hydrophilic material.

[0046] A linker is used to conjugate an immunoglobulin-binding protein, such as protein A, to a hydrophilic surface 115. Referring to Figure 1B, the conjugated particle 150 is shown. That is, particle 150 is the result of conjugating the immunoglobulin-binding protein 120 to the hydrophilic surface 115 using a linker. Any type of linker can be used as described above. Those skilled in the art will understand that there are various methods for attaching immunoglobulin-binding proteins to a hydrophilic layer. Many methods for conjugation are suitable for use with this technique. For example, an epoxy linker can be used to conjugate an immunoglobulin-binding protein to a hydrophilic base 115.

[0047] An immunoglobulin-binding protein provides an accessible binding site for immunoglobulins, i.e., antibodies, provided that the antibody contains a conserved region on which it binds to the immunoglobulin-binding protein. In one embodiment, the immunoglobulin-binding protein is protein A. In another embodiment, the immunoglobulin-binding protein is protein G. In yet another embodiment, the immunoglobulin-binding protein is protein A / G or protein L.

[0048] Most immunoglobulins (Ig) consist of four polypeptide chains: two identical heavy chains and two identical light chains linked by disulfide bonds. Within a given heavy or light chain, there are both variable and constant regions. The constant region, containing 2 to 4 constant domains (depending on the isotype), is highly conserved within a given isotype. Therefore, immunoglobulin-binding proteins that bind to a portion of the constant region are well-suited for capture by antibody affinity, regardless of the antibody target antigen.

[0049] Typically, immunoglobulin-binding proteins bind to the crystallizable fragment (Fc) region of an antibody, which includes a portion of the antibody's heavy chain constant domain. In at least one example, immunoglobulin-binding proteins have been shown to bind to the light chain constant domain instead.

[0050] Immunoglobulin-binding proteins suitable for use in this technology may exhibit strong binding affinity to the Fc portion of antibodies. This binding affinity can vary in strength depending on both isotype and species. For example, protein A exhibits strong binding affinity to the IgG isotype, but its binding affinity to IgA, IgD, IgE, and IgM isotypes is variable to nonexistent. Even within the IgG isotype, different subclasses may exhibit diverse binding affinities. Protein A has high binding affinity to human IgG1, IgG2, and IgG4, but very weak binding affinity to IgG3. On the other hand, protein A binds to mouse IgG3 but not to IgG1. Other examples of immunoglobulin-binding proteins, such as protein G, exhibit high binding affinity to all four subclasses of IgG. Methods for characterizing protein-protein interactions, including binding affinity across a range of environmental conditions, are well known in the art.

[0051] This technology enables the conjugation of any immunoglobulin-binding protein to the hydrophilic surface of non-porous polymer particles. Therefore, it is within the scope of this technology that immunoglobulin-binding proteins can bind to IgA, IgD, IgE, IgG, and / or IgM antibodies with varying levels of affinity. In some embodiments, the immunoglobulin-binding protein is selected from the group consisting of protein A, protein G, protein A / G, and protein L. In some embodiments, the immunoglobulin-binding protein may be a variant of protein A, protein G, protein A / G, or protein L. In some embodiments, the immunoglobulin-binding protein may have 80-85%, 85-90%, 90-95%, or 95-100% sequence identity with protein A, protein G, protein A / G, or protein L.

[0052] In some embodiments, the immunoglobulin-binding protein has a surface coverage of 3 to 9 μg per mg of particle. In some embodiments, the immunoglobulin-binding protein has a surface coverage of 3 to 3.5 μg, 3.5 to 4.0 μg, 4.0 to 4.5 μg, 4.5 to 5 μg, 5 to 5.5 μg, 5.5 to 6 μg, 6 to 6.5 μg, 6.5 to 7 μg, 7 to 7.5 μg, 7.5 to 8 μg, 8 to 8.5 μg, or 8.5 to 9 μg per mg of particle. In some embodiments, the immunoglobulin-binding protein is protein A and has a surface coverage of 3 to 9 μg per mg of particle. In some embodiments, the immunoglobulin-binding protein is protein G and has a surface coverage of 3 to 9 μg per mg of particle. In some embodiments, the immunoglobulin-binding protein is protein A / G, with a surface coverage of 3-9 μg per mg of particle. In some embodiments, the immunoglobulin-binding protein is protein L, with a surface coverage of 3-9 μg per mg of particle.

[0053] Examples 1 and 2 provide representative methods for synthesizing non-porous polymer particles that can be functionalized with immunoglobulin-binding proteins such as protein A, protein G, protein A / G, or protein L.

[0054] Without being bound by any particular theory, it is understood that the binding affinity of an antibody to an immunoglobulin-binding protein is at least partially dependent on the pH of the atmosphere. Therefore, the binding of the antibody to the immunoglobulin-binding protein is reversible by changing the pH of the buffer in the column of this technology.

[0055] Columns and devices In this technology, the above material is typically packed into a chromatography device, such as a chromatography column. The chromatography device includes a column body made of a metal or metal alloy, such as titanium or stainless steel. The column body contains multiple particles, such as the particles shown in Figure 1B.

[0056] Referring to Figure 2A, a chromatography column 205 is shown. This chromatography column 205 has a stainless steel column body 210. In Figure 2A, a portion 220 of the column body has been removed to show the locations of several particles 225. Figure 2B shows a cross-sectional view of the chromatography column 205 taken along line BB in Figure 2A.

[0057] The cross-sectional view in Figure 2B shows the position of the column body 210 surrounding and containing multiple particles 225. In some embodiments, alkylsilyl coatings or other high-performance surfaces are provided to limit or reduce the nonspecific binding of the sample to the walls or inner surface 230 of the column body 210. While we do not wish to be bound by theory, it is believed that alkylsilyl coatings covering metal surfaces prevent or minimize contact between the fluid passing through the column body 210 and the inner surface 230. Alkylsilyl coatings can be applied to the inner surface 230 of the metal column body 210, which defines what is called the wetting passage of the column. The metal wetting passage encompasses all surfaces formed from metal that are exposed to the fluid during the operation of the chromatography column. The metal wetting passage includes not only the walls of the column body but also the metal frit placed within the column. In some embodiments, the alkylsilyl coating is applied not only to the walls of the column body 210 but also to the frit.

[0058] Generally, alkylsilyl coatings are applied by vapor deposition. Vaporized precursors are placed in a reactor containing the area to be coated. These vaporized precursors react on the surface of the area to be coated to form a first layer of deposited material. Vapor deposition can be applied in a stepwise manner to increase the coating thickness by applying several layers of the deposited material to the surface, and / or to form a coating by applying layers of different materials (e.g., alternating the first and second materials).

[0059] In some embodiments, alkylsilyl coatings are applied to other parts of the liquid chromatography system. For example, alkylsilyl coatings can be applied to metal components located upstream and downstream of the column. In particular, alkylsilyl coatings can be applied to the injector and post-column piping and coupling devices of the liquid chromatography system (e.g., piping and coupling devices leading downstream components from the column, e.g., to the detector). Furthermore, affinity chromatography columns of this technology do not require the addition of additional organic modifiers to reduce nonspecific binding. Typically, the addition of organic modifiers (e.g., acetonitrile) is sometimes necessary in conjunction with adsorbents used in affinity chromatography to reduce nonspecific binding. Due to the already low nonspecific binding of the columns of this technology, organic modifiers are not required.

[0060] In one embodiment, the alkylsilyl coating comprises a hydrophilic, nonionic layer of polyethylene glycol silane. In another embodiment, the alkylsilyl coating is formed from one or more of the following precursor materials: bis(trichlorosilyl)ethane or bis(trimethoxysilyl)ethane. Other embodiments of alkylsilyl coatings suitable for use with the present technology are described in U.S. Patent Application Publication 2019 / 0086371 and U.S. Patent Application Publication 2022 / 0118443.

[0061] The chromatography device of this technology can be of a suitable size for use in HPLC, UHPLC, or FPLC (High-Performance Protein Liquid Chromatography) systems. For example, in embodiments where the multiple particles packed into the column have an average particle size of 3.5 micrometers or 1.7 micrometers, the column body of this technology can have an inner diameter of 1 mm to 4.6 mm and a column length of 5 to 50 cm. In certain embodiments, the column body has an inner diameter of 1 mm to 2.1 mm and a length of 15 to 50 cm.

[0062] How to use affinity chromatography columns The affinity chromatography column obtained using this technology can be used to capture antibodies that bind to specific immunoglobulin-binding proteins conjugated to particles in Figure 1B, based on their affinity. The affinity chromatography column is suitable for use in conjunction with any of the liquid chromatography systems described above. In some embodiments, the liquid chromatography system is an HPLC, UHPLC, or FPLC system.

[0063] Figure 3 provides an overview of a method (300) for performing antibody affinity-based capture using a chromatography column (305) with multiple particles functionalized with immunoglobulin-binding protein (225). The column (305) is connected to a liquid chromatography system (310), and a solution containing the antibody (315) is flowed through the column. Due to the binding affinity of the antibody to the immunoglobulin-binding protein conjugated to the particles (320), the antibody is immobilized on the column. This can be reversed by changing the atmosphere to reduce the binding affinity, for example, by making the pH acidic. In this way, the antibody is no longer immobilized and elutes from the column.

[0064] The examples described below use three comparison columns, referred to as comparison column 1, comparison column 2, and comparison column 3 throughout this specification.

[0065] Comparison column 1 is a polymer-based Protein A monolith in a 5.6 × 5 mm stainless steel column.

[0066] Comparative column 2 contains protein A conjugated to 12-micron non-porous polystyrene / divinylbenzene (PS / DVB) particles in a 4 × 35 mm polyether ether ketone column.

[0067] Comparative column 3 contains protein A conjugated to 20-micron, well-porous (500-1000 angstrom pores) polystyrene / divinylbenzene (PS / DVB) particles in a 2.1 × 30 mm polyether ether ketone column.

[0068] Example 3 describes a method for performing an affinity capture assay using an affinity chromatography column prepared with Protein A-functionalized particles. The column was used to bind and elute purified IgG at a range of concentrations. This was then compared to a comparative column 1 using similar conditions and samples.

[0069] Figure 4A shows a comparison of peak volumes for 0.25–20 μg of IgG compared to the experiment described in Example 3. Figure 4B shows a comparison of peak volumes for 0.25–300 μg of IgG compared to the experiment described in Example 3. The affinity chromatography column of this technology provides better peak volumes and enables more robust titration calculations compared to comparison column 1.

[0070] Example 4 describes a method for performing an affinity capture assay using an affinity chromatography column prepared with Protein A-functionalized particles. The column was used to bind and elute purified IgG at a range of concentrations. This was then compared to comparative columns 2 and 3 using similar samples and supplier-recommended protocols. Figure 5A demonstrates that the column of the present technology provides greatly improved peak shape and width compared to the comparative columns. Figure 5B shows that the column of the present technology results in a greatly reduced percentage carryover between affinity-based capture cycles. The affinity chromatography column of the present technology provided better peak volume compared to the comparative columns, enabling more robust titration calculations. The column of the present technology showed a sensitivity increase of >5X, and due to the increased sensitivity of the column, the detector reached saturation with an injection of 25 μg of IgG.

[0071] Furthermore, as described in Example 5, the column of this technology enables faster affinity-based capture of IgG than those available in the art without sacrificing sensitivity or performance. Figure 6A shows that the column of this technology enables affinity-based capture of IgG using a 1-minute binding phase that produces a peak area (Figure 6B) and carryover (Figure 6C) equivalent to a 3-minute binding phase. Thus, the column of this technology enables rapid determination of IgG titer using a binding phase time of approximately 1 minute.

[0072] Buffers suitable for use in liquid chromatography are well known in the art. A range of binding buffers are suitable for use with the disclosed techniques, and it is understood that those skilled in the art can determine a suitable binding buffer, i.e., a suitable binding buffer with a given antibody or sample, without excessive experimentation. In one embodiment, the binding buffer has a pH of 7.0 to 8.0. In some embodiments, the pH of the binding buffer is 7.0 to 7.1, 7.1 to 7.2, 7.2 to 7.3, 7.3 to 7.4, 7.4 to 7.5, 7.5 to 7.6, 7.6 to 7.7, 7.7 to 7.8, 7.8 to 7.9, or 7.9 to 8.0. In one embodiment, the elution buffer has a pH of 1.0 to 3.5. In some embodiments, the pH of the elution buffer is 1.0–1.2, 1.2–1.4, 1.4–1.6, 1.6–1.8, 1.8–2.0, 2.0–2.2, 2.2–2.4, 2.4–2.6, 2.6–2.8, 3.0–3.2, 3.2–3.4, or 3.4–3.5. It is important that the pH of the elution buffer is at least three times more acidic than that of the binding buffer.

[0073] In some embodiments, the binding buffer contains 20-100 mM sodium phosphate (pH 7.4) with 125 mM NaCl. In some embodiments, the binding buffer contains 100 mM sodium phosphate buffer (pH 7.4). In some embodiments, the binding buffer contains 20-200 mM ammonium acetate (pH 7.0-8.0). In some embodiments, the binding buffer contains 20-200 mM ammonium formate (pH 7.0-8.0). In some embodiments, the binding buffer is compatible with mass spectrometry.

[0074] In some embodiments, the elution buffer contains 20-50 mM sodium phosphate (pH 2.3). In some embodiments, the elution buffer contains 20-50 mM sodium phosphate (pH 2.3) with 100-500 mM NaCl. In some embodiments, the elution buffer contains 6 mM hydrochloric acid (pH 2.5). In some embodiments, the elution buffer contains 24 mM phosphate (pH 1.93-2.05). In some embodiments, the elution buffer contains 100 mM glycine (pH 2.5). In some embodiments, the elution buffer is 0.1%-2% acetic acid in water (pH 1.0-3.0). In some embodiments, the elution buffer is 0.1% formic acid in water (pH 1.0-3.0). In some embodiments, the elution buffer is compatible with mass spectrometry.

[0075] In some embodiments, the target sample is eluted from the column using stepwise elution, in which the mobile phase is switched from a binding buffer to an elution buffer. In some embodiments, the target sample is eluted from the column using gradient elution, in which the binding buffer is transitioned to the elution buffer as a gradient.

[0076] In one embodiment, the affinity chromatography column of this technology provides immunoglobulin elution with a small peak volume. In some embodiments, the peak volume is less than 50 μL. In some embodiments, the peak volume is less than 40 μL, less than 30 μL, or less than 20 μL. In some embodiments, the peak volume is less than 10 μL.

[0077] In one embodiment, the affinity chromatography column of this technology can be reused in an affinity capture assay. In this regard, after elution of antibody from the column, the column is then re-equilibriumized. Re-equilibriumization involves running a buffer through the column to remove any residual material from the previous assay. In some embodiments, re-equilibrium is performed using a binding buffer. In some embodiments, re-equilibrium is performed using an equilibration buffer. The equilibration buffer may have a pH less than or equal to that of the binding buffer. Due to the relationship between pH and the conformational stability and folding of proteins, it may be desirable to immediately neutralize the eluted sample with a base.

[0078] In one embodiment, the technology enables rapid re-equilibriumization of affinity chromatography columns. In some embodiments, affinity chromatography columns are re-equilibriumized for 1-10 minutes, 1-2, 2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10 minutes. In some embodiments, the chromatography column may require re-equilibriumization for a longer period of time.

[0079] After re-equilibriumization, the affinity chromatography column is immediately reused for affinity capture assays. The affinity chromatography column of this technology can be reused 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, or more than 200 times without loss of specificity or significant loss. In one embodiment, the affinity chromatography column of this technology can be stored between uses. Buffers suitable for storing affinity chromatography columns are well known in the art.

[0080] In one embodiment, the affinity chromatography column of this technology results in significantly reduced carryover of the applied sample after the re-equilibrium step.

[0081] Figures 4C and 5B show that the affinity chromatography column of this technology resulted in 2–5X less carryover between uses compared to comparative columns 1, 2, and 3.

[0082] Methods and Examples [Example 1] Addition of epoxy linker to hydrophilic particles Nonporous epoxy-modified hydrophilic particles used in the disclosed method were prepared as follows: In the first step, 1500 g of reagent alcohol (90% ethanol, about 5% methanol and about 5% isopropanol), 45.1 g of polyvinylpyrrolidone (PVP-40), 4.8 g of 2,2'-azobis(2-methylpropionitrile), 5.9 g of Triton® N-57, and 81.7 g of styrene were charged into the reactor. After the reactor was purged with nitrogen gas, the reaction mixture was heated and maintained at 70°C for 3 hours with stirring.

[0083] After 3 hours, a solution containing 110.4 g of divinylbenzene 80 (DVB), 39.7 g of PVP-40, 510 g of reagent alcohol, and 100.2 g of p-xylene was added to the reaction mixture at a constant flow rate over 2 hours. Following this step, a primer coating solution containing 26.0 g of glycidyl methacrylate (GMA), 26.0 g of ethylene glycol dimethacrylate (EDMA), 36.4 g of PVP-40, and 560 g of reagent alcohol was added to the reaction mixture at a constant flow rate over 1.5 hours.

[0084] After the reaction mixture was maintained at 70°C for a total of 20 hours, the particles were separated from the reaction slurry by filtration. The particles were then successively washed with methanol, tetrahydrofuran (THF), and acetone. The final product was dried in a vacuum oven at 45°C to obtain monodisperse 3.5 μm polymer particles. These particles contain a graded polystyrene / DVB core with a poly(GMA / EDMA) primer. While the above reaction conditions produce 3.5 μm polymer particles, it is understood that particles in the size range of 1.5 μm to 8 μm are within the scope of this disclosure. By varying the concentrations of PVP-40, 2,2'-azobis(2-methylpropionitrile), and Triton N-57, particle sizes within a range that can be produced by those skilled in the art can be produced.

[0085] The resulting 3.5 μm polystyrene / DVB particles with poly(GMA / EDMA) primers were then coated with a hydrophilic layer. 70 g of particles were hydrolyzed in 0.5 M H2SO4 at 60°C for 1–20 hours. The hydrolyzed particles were successively washed with Milli-Q water and methanol, and then dried overnight at 45°C in vacuum. The dried particles were added to a 1 L three-neck round-bottom flask equipped with an overhead stirring motor, stirring shaft and blades, a water-cooled condenser, a nitrogen inlet and a probe-controlled heating mantle. 700 mL of anhydrous diglyme (diethylene glycol dimethyl ether) was added, the flask was sealed, and purged with nitrogen for 15 minutes with gentle stirring. 2.0 g of potassium tert-butoxide was added, and the reaction was raised to 70°C. To generate a hydrophilic layer, a mixture of 10.5 g glycidol, 2.6 g glycerol triglycidyl ether, and 14.9 g anhydrous diglycimin was prepared separately and added to the particle mixture in four equal portions at 30-minute intervals. The reaction was maintained at 70°C for 20 hours, cooled to RT, and filtered. The resulting particles were washed six times with water and three times consecutively with methanol, and then dried overnight in vacuum at 45°C. As a result of the following procedure, a hydrophilic layer comprising 2-4% (by weight) of the total particles was obtained.

[0086] Twenty g of 3.5 μm particles with the resulting hydrophilic coating were added at room temperature to a mixture of 100 g of ethylene glycol diglycidyl ether (EGDGE) and 100 g of MeOH. One mL of 50% sodium hydroxide in water was added, and the reaction was stirred continuously for 20 hours. The particles were isolated by filtration, washed ten times with 400 mL of MeOH, and partially dried under a nitrogen stream. The particles were stored at 4°C in a methanol-moistened bed for later use. The resulting particles have a sufficient epoxide content to enable the functionalization of the particle surface.

[0087] [Example 2] Particles functionalized with Protein A The particles were prepared as described in Example 1 and functionalized with Protein A. 1.5 g of particles were mixed in 8.3 mL of 50 mM sodium phosphate buffer (pH 8). To this, 0.3 mL of a 50 mg / mL solution of Protein A (15 mg) was added. Next, 21.4 mL of sodium phosphate / ammonium sulfate solution was added dropwise. The reaction was then stirred for 20 hours at 24-37°C. The final concentration of ammonium sulfate and the reaction temperature can be adjusted to manipulate the degree of Protein A coating on the given particles.

[0088] After 20 hours of incubation, 1 g of ethanolamine was added to 5 mL of sodium phosphate buffer, and the reaction was stirred at RT for 3 hours. The particles were then isolated by filtration and washed three times with water (pH 4, adjusted with HCl), twice with water, and twice consecutively with storage buffer (20 mM PBS, pH 7.3, 0.02% sodium azide). The particles were stored as a slurry in storage buffer (approximately 10 mL buffer / g particle) in a sealed container at 4°C. The protein A coverage of the particles was determined using a standard bicinchoninic acid assay (BCA). The maximum surface coverage of protein A in the particles was determined to be 4.2–5.0 μg of protein A per mg of particle. Typical protein A particles have a surface coverage of approximately 4.8 μg of protein A per mg of particle (particle type A).

[0089] Alternatively, particles prepared as described in Example 1 can be functionalized with protein G. 1.5 g of particles are mixed in 8.3 mL of 50 mM sodium phosphate buffer (pH 8). To this, 0.3 mL of a 50 mg / mL solution of protein G (15 mg) can be added. Next, 21.4 mL of sodium phosphate / ammonium sulfate solution can be added dropwise, and the reaction can be stirred at 24-37°C for 20 hours. The final concentration of ammonium sulfate and the reaction temperature can be adjusted to manipulate the degree of protein G coating on the given particles. After 20 hours of incubation, 1 g of ethanolamine in 5 mL of sodium phosphate buffer can be added, and the reaction can be stirred at RT for 3 hours. The particles can be isolated by filtration and washed three times with water (pH 4, adjusted with HCl), twice with water, and twice consecutively with a storage buffer (e.g., 20 mM PBS, pH 7.3, 0.02% sodium azide). The protein G coating of particles can be determined using methods known in the art, such as standard BCA assays.

[0090] Alternatively, particles prepared as described in Example 1 can be functionalized with Protein A / G. 1.5 g of particles are mixed in 8.3 mL of 50 mM sodium phosphate buffer (pH 8). To this, 0.3 mL of a 50 mg / mL solution of Protein A / G (15 mg) can be added. Next, 21.4 mL of sodium phosphate / ammonium sulfate solution is added dropwise, and the reaction can be stirred at 24-37°C for 20 hours. The final concentration of ammonium sulfate and the reaction temperature can be adjusted to manipulate the degree of Protein A / G coating on the given particles. After 20 hours of incubation, 1 g of ethanolamine in 5 mL of sodium phosphate buffer is added, and the reaction can be stirred at RT for 3 hours. The particles are isolated by filtration and can be washed three times with water (pH 4, adjusted with HCl), twice with water, and twice consecutively with a storage buffer (e.g., 20 mM PBS, pH 7.3, 0.02% sodium azide). The protein A / G coating of particles can be determined using methods known in the art, such as standard BCA assays.

[0091] Alternatively, particles prepared as described in Example 1 can be functionalized with Protein L. 1.5 g of particles are mixed in 8.3 mL of 50 mM sodium phosphate buffer (pH 8). To this, 0.3 mL of a 50 mg / mL solution of Protein L (15 mg) can be added. Next, 21.4 mL of sodium phosphate / ammonium sulfate solution is added dropwise, and the reaction can be stirred at 24-37°C for 20 hours. The final concentration of ammonium sulfate and the reaction temperature can be adjusted to manipulate the degree of Protein L coating on the given particles. After 20 hours of incubation, 1 g of ethanolamine in 5 mL of sodium phosphate buffer is added, and the reaction can be stirred at RT for 3 hours. The particles are isolated by filtration and can be washed three times with water (pH 4, adjusted with HCl), twice with water, and twice consecutively with a storage buffer (e.g., 20 mM PBS, pH 7.3, 0.02% sodium azide). The protein L coating of particles can be determined using methods known in the art, such as standard BCA assays.

[0092] [Example 3] IgG binding using a protein A affinity chromatography column The particles prepared in Example 2 were used to perform IgG affinity-based capture. Protein A-functionalized particles were packed into 2.1 × 20 mm hardware. This was connected to a liquid chromatography apparatus and equilibrated with 20 mM sodium phosphate (pH 7.4) and 125 mM sodium chloride at a flow rate of 1 mL / min for 5 minutes.

[0093] Once equilibrated, 10 μL of 10 μg / mL IgG injection solution was injected into the column and flowed at a flow rate of 2 mL / min for 0.4 minutes to enable binding. The column effluent was monitored using a UV detector (280 nm). After 0.4 minutes of binding, elution buffer (20 mM sodium phosphate (pH 2.7)) was flowed through the column at a flow rate of 2 mL / min for 0.4 minutes. The column was then re-equilibriumated with 20 mM sodium phosphate (pH 7.4) and 125 mM sodium chloride at a flow rate of 2 mL / min for 0.4 minutes.

[0094] After re-equilibrating the column, 5 μL of 10 μg / mL IgG injection solution was injected into the column, and the binding, elution, and equilibration steps were repeated as described above. This was repeated using volumes that yielded total IgG concentrations of 0.5, 1, 3, 5, 10, 20, 50, 75, 100, 300, and 500 μg. For comparison, the process was carried out under similar conditions using comparison column 1.

[0095] The Protein A column of this technology (solid black line) produced an improved peak area compared to comparative column 1 (dotted line) (Figure 4A-B), enabling more robust antibody titer calculations. After the elution and equilibration steps, the Protein A column of this technology produced a significantly reduced (2-3X less) carryover of the target antibody compared to comparative column 1 (Figure 4C).

[0096] [Example 4] IgG binding in a protein A affinity chromatography column The particles prepared in Example 2 were used to perform IgG affinity-based capture. Protein A-functionalized particles were packed into 2.1 × 20 mm hardware. This was connected to a liquid chromatography apparatus and equilibrated with binding buffer (100 mM sodium phosphate (pH 7.4)) at a flow rate of 1 mL / min for 10 minutes. The binding buffer could be in the range of 20–100 mM sodium phosphate (pH 7.4), containing 125 mM sodium chloride or 100 mM sodium phosphate. Comparative column 3 (also referred to herein as particle type G) and comparative column 2 were used as a reference for comparison. Comparative column 3 was equilibrated with 20–100 mM sodium phosphate (pH 7.4) containing 120–150 mM NaCl. Comparative column 2 was equilibrated with 50 mM sodium phosphate (pH 7.4), 150 mM NaCl, and 5% acetonitrile.

[0097] Once equilibrated, 10 μL of 10 μg / mL IgG injection solution was injected into the column and flowed at a flow rate of 1 mL / min for 1 minute to enable binding. The column effluent was monitored using a UV detector (280 nm). After a 1 minute binding step, elution buffer (24 mM phosphate (pH 1.93)) was flowed through the column at a flow rate of 1 mL / min for 1 minute. The column was then re-equilibriumated with 100 mM sodium phosphate (pH 7.4) at a flow rate of 1 mL / min for 10 minutes.

[0098] After re-equilibriumating the column, 10 μL injection solutions of various IgG concentrations were injected into the column, and the binding, elution, and equilibration steps were repeated as described above. This process was carried out using various concentrations of IgG, resulting in total IgG concentrations of 0.5, 1, 2.5, 5, 10, and 25 μg. IgG concentrations of >25 μg caused UV detector saturation due to the increased sensitivity of the Protein A column.

[0099] For comparison, the process was performed using similar samples with comparison columns 2 and 3. For comparison column 3, 10 μL of 10 μg / mL IgG was injected into the column and flowed at a flow rate of 1 mL / min for 0.6 min. The column effluent was monitored using a UV detector (280 nm). After the binding step, elution buffer (50 mM sodium phosphate, pH 2.3) was flowed through the column at a flow rate of 1 mL / min for 1.2 min. The column was re-equilibriumized with the binding buffer for 2.4 min. For comparison column 2, 10 μL of 10 μg / mL IgG was injected into the column and flowed at a flow rate of 1 mL / min for 0.6 min. The column effluent was monitored using a UV detector (280 nm). After the binding step, elution buffer (50 mM sodium phosphate, pH 2.3, 150 mM NaCl, 5% acetonitrile) was flowed through the column at a flow rate of 1 mL / min for 1.2 min. The column was re-equilibriumized with the binding buffer for 2.4 min. This was repeated using injection volumes that produced total IgG amounts of 0.5, 1, 2.5, 5, 10, 25, 50, 75, and 100 ug of IgG. Unlike the column in this technology, no UV detector saturation was observed in comparative columns 2 and 3, even at the highest concentrations tested.

[0100] The Protein A column of this technology (black dashed line) produced improved peak shape and width (i.e., peak area) compared to comparative column 3 (dashed line) and comparative column 2 (black solid line) (Figure 5A), enabling more robust antibody titer calculations. The Protein A column of this technology produced a significantly reduced (one order of magnitude) carryover of target antibody compared to the comparative column after the elution and equilibration steps (Figure 5B). Furthermore, the Protein A column of this technology produced a significantly increased sensitivity (>5X) compared to the comparative column, and showed a one-order-of-magnitude reduction in peak width after the elution and equilibration steps compared to comparative column 3.

[0101] [Example 5] Protein A column enables rapid capture of IgG due to its affinity. The particles prepared in Example 2 were used to perform IgG affinity-based capture. Protein A-functionalized particles were packed into 2.1 × 20 mm hardware. This was connected to a liquid chromatography apparatus and equilibrated with binding buffer (100 mM sodium phosphate (pH 7.4)) at a flow rate of 0.5 mL / min for 10 minutes. Once equilibrated, 10 μL of 10 μg / μL IgG injection solution was injected into the column and flowed at a flow rate of 1 mL / min for 1 minute to enable binding. The column effluent was monitored using a UV detector (280 nm). The captured IgG was eluted with elution buffer (100 mM glycine (pH 2.5)) flowed through the column at a flow rate of 1 mL / min for 0.5 minutes. After this, the column was re-equilibriumized with binding buffer flowed at a flow rate of 1 mL / min for 0.5 minutes. After re-equilibriumizing the column, 10 μL of injection solution of various concentrations of IgG was injected into the column, and the binding, elution, and equilibration steps were repeated as described above. This was performed using concentrations that yielded total IgG amounts of 0.5, 1, 2.5, 5, 10, and 25 μg. For comparison, these procedures were also repeated using a binding step in which IgG was injected into the column and flowed at a flow rate of 1 mL / min for 3 minutes to enable binding.

[0102] As shown in Figure 6A, both the 1-minute (solid black line) and 3-minute (dashed black line) binding phases produced similar peak widths and sensitivities. As shown in Figures 6B-6C, the peak area (Figure 6B) and carryover (Figure 6C) were similar across the different binding phase times tested. Therefore, the Protein A column of this technology can efficiently capture IgG using a 1-minute binding phase.

[0103] [Example 6] Material functionalized with additional protein A Non-porous silica particles with hybrid coating, diol coating, and hydrophilic coating - Particle type B Silica-based, nonporous, epoxy-modified hydrophilic particles were prepared. Nonporous 3.6-micron silica particles were heat-treated in air for 10 hours (900°C). The surface of the particles was rehydroxylated with 10% v / v nitric acid at 100°C for 16 hours. The reaction was then cooled to below 40°C, and the particles were isolated by filtration. The product was washed with water until the pH of the filtrate was above 5, and then washed three times with acetone. The isolated particles were dried overnight in a vacuum oven at 80°C.

[0104] Hydroxylated silica particles were coated with 1,2-bis(triethoxysilane)ethane (BTEE) containing tetraethyl orthosilicate (TEOS). The silane reagent was initially prepared from the incomplete (approximately 68%) hydrolysis condensation of BTEE containing tetraethyl orthosilicate (TEOS). Ethanol (3.1 mol ethanol / mol silane), TEOS (in a 1:4 molar ratio with BTEE), and 0.1 M HCl (19.7 g / mol silane reagent) were added to the BTEE. The solution was heated at 70°C for 18 hours under an inert atmosphere. The reaction was increased to 90°C to remove the ethanol. The reaction was further increased to 100°C for 1 hour under an inert atmosphere. The mixture was cooled to room temperature (RT) to obtain the condensation product.

[0105] Silica particles were completely dispersed in toluene (21 mL / g particles). Residual water was removed from the material by azeotropic distillation (110°C, 1 hour). The reaction was maintained at 40°C while silane reagent (0.821 g / g particles) was added and stirred for 10 minutes. The catalyst aqueous NH4OH was added (0.05 g / g particles) and stirred for a further 10 minutes at 40°C, and then for an additional 2 hours at 60°C. The reaction was cooled to RT, and the particles were isolated by filtration. The particles were washed twice with ethanol (10 mL / g) and then dispersed in 10 mL of 70:30 water:ethanol (v / v) per gram of particles. Ammonium hydroxide solution (1 g / g particles) was added, and the mixture was stirred at 50°C for 2 hours. The reaction was cooled to below 40°C, and the particles were isolated by filtration. The isolated particles were washed twice with 10 mL of 1:1 methanol:water (v / v) per gram of particles, and then twice with methanol. The surface-modified particles were dried in a vacuum at 70°C for 16 hours.

[0106] To ensure uniformity of the hybrid coating layer, the modified particles were subjected to a hot water treatment process followed by heating (100-140°C) and increased pH (8-9.8) according to the methods described herein by reference: U.S. Patents 6,686,035, 7,223,473, and 7,919,177, and International Publication No. 2008 / 103423.

[0107] The modified particles were dispersed in 1 M HCl (8.4 mL / g particles), and the mixture was stirred at 100°C for 20 hours. The reaction was cooled to below 40°C, and the particles were isolated by filtration. The isolated particles were washed with water until the pH of the filtrate was above 5, and then the particles were washed three times with acetone. The isolated surface-modified particles were dried in a vacuum at 70°C for 16 hours.

[0108] Next, the resulting 3.6 micron particles were coated with glycidoxypropyltrimethoxysilane (GPTMS). Approximately 5 mL / gram of 20 mM sodium acetate buffer (pH 5.5) was added to a round-bottom flask equipped with a thermometer, condenser, and mechanical stirrer. After the solution was heated to 70°C, the GPTMS was mixed to a concentration of 10.22 μmol / m³. 2 The solution was added to the flask at the specified concentration and mixed at the specified temperature. After 1 hour, the particles were added to the flask (1 g particles / 5 mL buffer solution). The mixture was stirred at 70°C for 20 hours, after which the reaction was cooled to below 40°C and filtered. The product was washed three times with water and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirrer. Acetic acid (0.1 M) was added to the flask (5 mL / gram particles), and the slurry was heated at 70°C for 20 hours. The flask was cooled to below 40°C and filtered. The product was washed with water until the pH of the supernatant was above 5, and then washed three times with methanol. The isolated particles were dried in a vacuum oven at 70°C for 16 hours.

[0109] Ten grams of the resulting particles were added to a 1 L three-necked round-bottom flask equipped with an overhead stirring motor, stirring shift, stirring blades, a water-cooled condenser, a nitrogen inlet, and a probe-controlled heating mantle. 100 mL of anhydrous diglycylm (diethylene glycol dimethyl ether) was added. The flask was sealed and purged with nitrogen for 15 minutes with gentle stirring. 0.45 g of potassium tert-butoxide was added, and the reaction was raised to 70°C. To create a hydrophilic layer, a mixture of 1.5 g of glycidol, 0.4 g of glycerol triglycidyl ether, and 2.2 g of anhydrous diglycylm was prepared separately and added to the particle mixture in four equal portions with 30-minute intervals. The reaction was held at 70°C for 20 hours, cooled to RT, and filtered. The resulting particles were washed six times with water and three times with methanol in succession, and dried overnight at 45°C in vacuum. This procedure produced a hydrophilic layer of 2–4% by weight of the total particles.

[0110] 5 g of 3.6 micron particles with the obtained hydrophilic coating were added RT-to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol. 0.25 mL of 50% sodium hydroxide in water was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed 10 times with methanol, and partially dried under a nitrogen stream. The particles were stored at 4°C on a methanol-moistened bed. The resulting particles have a sufficient epoxide content to enable the functionalization of the particle surface.

[0111] The particles were functionalized with protein A as described in Example 2, yielding approximately 8.21 ug of protein A per mg of particle.

[0112] The particles were tested for their ability to bind and elute monoclonal antibodies (NISTmAb standards available from Sigma-Aldrich). The particles were packed into a 2.1 × 200 mm column and connected to an ultra-high-performance liquid chromatography system (Acquity Premier, available from Waters Technologies Corporation, Milford MA). The resulting column was rapidly flushed with water (pH 4) at 0.5 mL / min for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. 10 μL injection solutions of monoclonal antibody standards at various concentrations (ranging from 0.001 to 1 μg / uL) were injected into the column using 0.1 M sodium phosphate (pH 7.4) buffer at a flow rate of 1 mL / min. A blank injection solution of binding buffer was used between samples to calculate carryover. Elution was performed for 1 minute using either 24 mM phosphate (pH 2) or 100 mM glycine (pH 2.4). The column was equilibrated for 1 minute using 0.1 M sodium phosphate buffer (pH 7.4).

[0113] As shown in Figure 7A (24 mM phosphate (pH 2)) and Figure 7B (100 mM glycine (pH 2.4)), poor peak shape, fronting, and shoulders were observed due to protein A leaking from the column. Significant peak tailing was observed for both conditions due to nonspecific binding of the monoclonal antibody sample. Thus, even with coating of the silica surface with hybrid materials, diols, and hydrophilic layers, high nonspecific binding due to silica particles was still observed. Low detection amounts of 0.025 ug and 0.05 ug were observed for phosphate and glycine elution buffers, respectively. At low concentrations, >40% carryover was observed due to leaking protein A. At higher concentrations, >6% carryover was observed. Figure 7C shows the linear correlation between monoclonal antibody concentration and peak area for both elution methods.

[0114] Non-porous silica particles (particle type D) with hybrid coating and diol coating. Silica-based, nonporous, epoxy-modified hydrophilic particles were prepared. Nonporous 3.6-micron silica particles were heat-treated in air for 10 hours (900°C). The surface of the particles was rehydroxylated with 10% v / v nitric acid at 100°C for 16 hours. The reaction was then cooled to below 40°C, and the particles were isolated by filtration. The product was washed with water until the pH of the filtrate was above 5, and then washed three times with acetone. The isolated particles were dried overnight in a vacuum oven at 80°C.

[0115] Hydroxylated silica particles were coated with 1,2-bis(triethoxysilane)ethane (BTEE) containing tetraethyl orthosilicate (TEOS). The silane reagent was initially prepared from the incomplete (approximately 68%) hydrolysis condensation of BTEE containing tetraethyl orthosilicate (TEOS). Ethanol (3.1 mol ethanol / mol silane), TEOS (in a 1:4 molar ratio with BTEE), and 0.1 M HCl (19.7 g / mol silane reagent) were added to the BTEE. The solution was heated at 70°C for 18 hours under an inert atmosphere. The reaction was increased to 90°C to remove the ethanol. The reaction was further increased to 100°C for 1 hour under an inert atmosphere. The mixture was cooled to room temperature (RT) to obtain the condensation product.

[0116] Silica particles were completely dispersed in toluene (21 mL / g particles). Residual water was removed from the material by azeotropic distillation (110°C, 1 hour). The reaction was maintained at 40°C while silane reagent (0.821 g / g particles) was added and stirred for 10 minutes. The catalyst aqueous NH4OH was added (0.05 g / g particles) and stirred at 40°C for an additional 10 minutes, and then at 60°C for an additional 2 hours. The reaction was cooled to RT, and the particles were isolated by filtration. The particles were washed twice with ethanol (10 mL / g) and then dispersed in 10 mL of 70:30 water:ethanol (v / v) per gram of particles. Ammonium hydroxide solution (1 g / g particles) was added, and the mixture was stirred at 50°C for 2 hours. The reaction was cooled to below 40°C, and the particles were isolated by filtration. The isolated particles were washed twice with 10 mL of 1:1 methanol:water (v / v) per gram of particles, and then twice with methanol. The surface-modified particles were dried under vacuum at 70°C for 16 hours.

[0117] To ensure uniformity of the hybrid coating layer, the modified particles were subjected to a hot water treatment process followed by increased heating (100-140°C) and increased pH (8-9.8) according to the methods described herein by reference: U.S. Patents No. 6,686,035, No. 7,223,473, and No. 7,919,177 and International Publication No. 2008 / 103423.

[0118] The modified particles were dispersed in 1 M HCl (8.4 mL / g particles), and the mixture was stirred at 100°C for 20 hours. The reaction was cooled to below 40°C, and the particles were isolated by filtration. The isolated particles were washed with water until the pH of the filtrate was above 5, and then the particles were washed three times with acetone. The isolated surface-modified particles were dried under vacuum at 70°C for 16 hours.

[0119] The resulting 3.6 micron particles were then coated with glycidoxypropyltrimethoxysilane (GPTMS). Approximately 5 mL / gram of 20 mM sodium acetate buffer (pH 5.5) was added to a round-bottom flask equipped with a thermometer, condenser, and mechanical stirrer. After the solution was heated to 70°C, the GPTMS was mixed to a concentration of 10.22 μmol / m³. 2 The mixture was added to a flask at the specified concentration and mixed at the specified temperature. After 1 hour, the particles were added to the flask (1 g particles / 5 mL buffer solution). The mixture was stirred at 70°C for 20 hours, after which the reaction was cooled to below 40°C and filtered. The product was washed three times with water and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirrer. Acetic acid (0.1 M) was added to the flask (5 mL / gram particles), and the slurry was heated at 70°C for 20 hours. The flask was cooled to below 40°C and filtered. The product was washed with water until the pH of the supernatant rose above 5, and then washed three times with methanol. The isolated particles were dried in a vacuum oven at 70°C for 16 hours.

[0120] 5 g of the resulting 3.6 micron particles with a hydrophilic coating were added RT-to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol. 0.25 mL of 50% sodium hydroxide in water was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed 10 times with methanol, and partially dried under a nitrogen stream. The particles were stored at 4°C on a methanol-moistened bed. The resulting particles have a sufficient epoxide content to enable functionalization of the particle surface.

[0121] The particles were functionalized with protein A as described in Example 2, yielding approximately 1.84 ug of protein A per mg of particle.

[0122] The ability of particles to bind and elute monoclonal antibodies (NISTmAb standards, available from Sigma-Aldrich) was tested. The particles were packed into a 2.1 × 200 mm column and connected to an ultra-high-performance liquid chromatography system (Acquity Premier, Waters Technologies Corporation, available from Milford MA). The column was rapidly flushed with water (pH 4) at 0.5 mL / min for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. 10 μL injection solutions of monoclonal antibody standards at various concentrations (ranging from 0.001 to 1 μg / uL) were injected into the column using 0.1 M sodium phosphate buffer (pH 7.4) at a flow rate of 1 mL / min. Blank injection solutions of binding buffer were used between samples to calculate carryover. Elution was performed for 1 minute with either 24 mM phosphate (pH 2) or 100 mM glycine (pH 2.4). The column was equilibrated for 1 minute using 0.1 M sodium phosphate buffer (pH 7.4).

[0123] The detection limit was 0.01 ug for the phosphoric acid elution method and 0.25 ug for the glycine elution method. Retention time was shifted in the glycine elution method (see Figure 8B) compared to the phosphoric acid elution method (see Figure 8A). The maximum applicable pressure was 2950 psi. Figure 8C shows the linear correlation between monoclonal antibody concentration and mean peak area for both elution methods. Carryover was less than 3% for glycine elution and <22% for phosphoric acid elution. The lack of a hydrophilic layer leads to tailing due to nonspecific binding of the monoclonal antibody to the particle surface.

[0124] Non-porous silica particles with diol coating (particle type H) Silica-based, nonporous, epoxy-modified hydrophilic particles were prepared. Nonporous 3.6-micron silica particles were heat-treated in air for 10 hours (900°C). The surface of the particles was rehydroxylated with 10% v / v nitric acid at 100°C for 16 hours. The reaction was then cooled to below 40°C, and the particles were isolated by filtration. The product was washed with water until the pH of the filtrate was above 5, followed by three washes with acetone. The isolated particles were dried overnight in a vacuum oven at 80°C.

[0125] The resulting 3.6 micron particles were then coated with glycidoxypropyltrimethoxysilane (GPTMS). 20 mM sodium acetate buffer (pH 5.5) was added to a round-bottom flask equipped with a thermometer, condenser, and mechanical stirrer at a concentration of approximately 5 mL / gram of particles. The solution was heated to 70°C, after which GPTMS was added at a concentration of 10.22 μmol / m³. 2 The solution was added to the flask at the specified concentration and mixed at the specified temperature. After 1 hour, the particles were added to the flask (1 g particles / 5 mL buffer solution). The mixture was stirred at 70°C for 20 hours, after which the reaction was cooled to below 40°C and filtered. The product was washed three times with water and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirrer. Acetic acid (0.1 M) was added to the flask (5 mL / gram particles), and the slurry was heated at 70°C for 20 hours. The flask was cooled to below 40°C and filtered. The product was washed with water until the pH of the supernatant rose above 5, and then washed three times with methanol. The isolated particles were dried in a vacuum oven at 70°C for 16 hours.

[0126] 5 g of 3.6 micron particles with a hydrophilic coating were added RT-to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol. 0.25 mL of 50% sodium hydroxide in water was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed 10 times with methanol, and partially dried under a nitrogen stream. The particles were stored at 4°C on a methanol-moistened bed. The resulting particles have a sufficient epoxide content to enable functionalization of the particle surface.

[0127] The particles were functionalized with protein A as described in Example 2, yielding approximately 2.7 ug of protein A per mg of particle.

[0128] The ability of particles to bind and elute monoclonal antibodies (NISTmAb standards, available from Sigma-Aldrich) was tested. The particles were packed into a 2.1 × 200 mm column and connected to an ultra-high-performance liquid chromatography system (Acquity Premier, Waters Technologies Corporation, available from Milford MA). The column was rapidly flushed with water (pH 4) at 0.5 mL / min for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. 10 μL injection solutions of monoclonal antibody standards at various concentrations (ranging from 0.001 to 1 μg / uL) were injected into the column at a flow rate of 1 mL / min using 0.1 M sodium phosphate (pH 7.4) buffer. Blank injection solutions of binding buffer were used between samples to calculate carryover. Elution was performed for 1 minute with either 24 mM phosphate (pH 2) or 100 mM glycine (pH 2.4). The column was equilibrated for 1 minute using 0.1 M sodium phosphate buffer (pH 7.4).

[0129] Significant peak tailing was observed with both elution methods, as shown in Figure 9A (24 mM phosphate (pH 2)) and Figure 9B (100 mM glycine (pH 2.4)). Peak splitting was observed from 0.25 ug / uL with phosphate elution and from 1 ug / uL with glycine elution. The maximum applicable pressure was 2500 psi. Figure 9C shows a linear correlation between monoclonal antibody concentration and mean peak area for the glycine elution method. Carryover was >3% for both elution conditions. The lack of a hydrophilic layer results in distorted peaks and significant tailing due to nonspecific binding of the monoclonal antibody to the particle surface.

[0130] Non-porous silica particles (particle type E) with diol coating and hydrophilic coating. Silica-based, nonporous, epoxy-modified hydrophilic particles were prepared. Nonporous 3.6-micron silica particles were heat-treated in air for 10 hours (900°C). The surface of the particles was rehydroxylated with 10% v / v nitric acid at 100°C for 16 hours. The reaction was then cooled to below 40°C, and the particles were isolated by filtration. The product was washed with water until the pH of the filtrate was above 5, followed by three washes with acetone. The isolated particles were dried overnight in a vacuum oven at 80°C.

[0131] The resulting 3.6 micron particles were then coated with glycidoxypropyltrimethoxysilane (GPTMS). Approximately 5 mL / gram of 20 mM sodium acetate buffer (pH 5.5) was added to a round-bottom flask equipped with a thermometer, condenser, and mechanical stirrer. After the solution was heated to 70°C, GPTMS was added at a concentration of 10.22 μmol / m³. 2 The mixture was added to a flask at the specified concentration and mixed at the specified temperature. After 1 hour, the particles were added to the flask (1 g particles / 5 mL buffer solution). The mixture was stirred at 70°C for 20 hours, after which the reaction was cooled to below 40°C and filtered. The product was washed three times with water and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirrer. Acetic acid (0.1 M) was added to the flask (5 mL / gram particles), and the slurry was heated at 70°C for 20 hours. The flask was cooled to below 40°C and filtered. The product was washed with water until the pH of the supernatant rose above 5, and then washed three times with methanol. The isolated particles were dried in a vacuum oven at 70°C for 16 hours.

[0132] Ten grams of the obtained particles were added to a 1 L three-necked round-bottom flask equipped with an overhead stirring motor, stirring shift, stirring blades, a water-cooled condenser, a nitrogen inlet, and a probe-controlled heating mantle. 100 mL of anhydrous diglycylm (diethylene glycol dimethyl ether) was added. The flask was sealed and purged with nitrogen for 15 minutes with gentle stirring. 0.45 g of potassium tert-butoxide was added, and the reaction was raised to 70°C. To produce a hydrophilic layer, a mixture of 1.5 g glycidol, 0.4 g glycerol triglycidyl ether, and 2.2 g anhydrous diglycylm was prepared separately and added to the particle mixture in four equal portions at 30-minute intervals. The reaction was maintained at 70°C for 20 hours, cooled to RT, and filtered. The obtained particles were washed six times with water and three times consecutively with methanol, and dried overnight at 45°C under vacuum. The procedure yielded a hydrophilic layer of 2–4% by weight of the total particles.

[0133] 5 g of 3.6 micron particles with a hydrophilic coating were added RT-to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol. 0.25 mL of 50% sodium hydroxide in water was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed 10 times with methanol, and partially dried under a nitrogen stream. The particles were stored at 4°C on a methanol-moistened bed. The resulting particles have a sufficient epoxide content to enable the functionalization of the particle surface.

[0134] The particles were functionalized with protein A as described in Example 2, yielding approximately 1.47 ug of protein A per mg of particle.

[0135] The ability of particles to bind to and elute monoclonal antibodies (NISTmAb standards, available from Sigma-Aldrich) was tested. The particles were packed into a 2.1 × 200 mm column and connected to an ultra-high-performance liquid chromatography system (Acquity Premier, Waters Technologies Corporation, available from Milford MA). The column was rapidly flushed with water (pH 4) at 0.5 mL / min for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. 10 μL injection solutions of monoclonal antibody standards at various concentrations (ranging from 0.001 to 1 μg / uL) were injected into the column at a flow rate of 1 mL / min using 0.1 M sodium phosphate (pH 7.4) buffer. Blank injection solutions of binding buffer were used between samples to calculate carryover. Elution was performed for 1 minute using either 24 mM phosphate (pH 2) or 100 mM glycine (pH 2.4). The column was equilibrated for 1 minute using 0.1 M sodium phosphate buffer (pH 7.4).

[0136] The detection limits were 0.01 ug for the phosphate elution method and 0.25 ug for the glycine elution method. Retention times were shifted between the glycine elution method (see Figure 10B) and the phosphate elution method (see Figure 10A). The maximum applicable pressure was 3100 psi. Figure 10C shows the linear correlation between monoclonal antibody concentration and mean peak area for both elution methods. Carryover was less than 7% for both elution methods. The very low amount of protein A immobilized on the surface indicates that less epoxide was available for the protein A conjugate. High carryover and considerable tailing indicate that the hydrophilic layer was not optimal.

[0137] In general, silica-based materials are not ideal for affinity chromatography methods due to the tendency of silica to have high levels of nonspecific bonding. The particle types above (B, D, H, and E) indicate that shielding the silica surface by a layer of hybrid material, a layer of diol, and / or a hydrophilic coating was insufficient to eliminate nonspecific bonding. While we do not wish to be bound by any particular theory, silica-based particle types may also have lower protein A conjugate efficiency, which can lead to high leakage.

[0138] Non-porous polymer particles (particle type C) having a diol coating and a hydrophilic coating. Polymer-based nonporous particles were prepared. 65.1 grams of polyvinylpyrrolidone (PVP-40), 8.9 grams of Triton N-57, 2452.6 grams of reagent alcohol (90% ethanol, 5% methanol, 5% isopropanol), and 153.3 grams of p-xylene were charged into a 4 L cylinder flask reactor equipped with mechanical stirring, a condenser, and a thermocouple. The reaction was carried out with stirring at 200 rpm, and dissolved oxygen in the solution was removed by subsurface purging with nitrogen. After the dissolved oxygen level was reduced to less than 1 ppm, 2.1 grams of azobisisobutyronitrile and 105 grams of divinylbenzene were added to the reaction. The reaction was raised to 70°C and maintained for 20 hours.

[0139] After 1.8 grams of azobisisobutyronitrile was added to the reaction, 67.6 grams of divinylbenzene (DVB) and a solution of 20.3 grams of PVP-40 in 202.7 grams of reagent alcohol were added via a pump at a constant flow rate over 120 minutes. The reaction was continued at 70°C for at least 12 hours. The particles were separated from the reaction slurry by filtration and washed sequentially with methanol, tetrahydrofuran (THF), and acetone. The final product was dried overnight in a vacuum oven at 45°C.

[0140] A 46.2 - gram polymer core was dispersed in 231 grams of ethanol by ultrasonic treatment for 4 minutes using an ultrasonic horn and transferred to a round - bottom flask equipped with a mechanical - grade stirrer, condenser, and thermocouple. 49.1 grams of ammonium hydroxide and 462 grams of toluene were added to the reaction and mixed for 60 minutes at RT. The reaction was raised to 35 °C, and a mixture of 26.06 grams of TEOS and 66.34 grams of BTEE diluted with 148.9 grams of ethanol and 148.9 grams of toluene was charged via a peristaltic pump at a constant flow rate of 0.95 mL / min. After the reaction, the particles were separated by filtration, washed 6 times with methanol, and dried overnight at 45 °C in a vacuum oven.

[0141] The obtained polymer - core particles were dispersed in an aqueous solution of 0.3 M tris(hydroxymethyl)aminomethane at a slurry concentration of 5 mL / g. The pH was adjusted to 9.8 using acetic acid. Then the slurry was sealed in a stainless - steel autoclave and heated to 155 °C for 20 hours. After cooling to RT, the product was isolated on a 0.5 - micron filter paper and washed with water and methanol. The particles were dried at 80 °C under vacuum for 16 hours.

[0142] Following the hot - water step, 50 g of the particles were refluxed in 1 M hydrochloric acid at a slurry concentration of 10 mL / g for 10 hours. The reaction was cooled to RT and the particles were isolated by filtration. The particles were washed with water until the pH of the filtrate was higher than 5. The semi - neutralized particles were washed 3 times with acetone (10 mL / g). The obtained particles were sized to remove aggregates, and a final particle size of 2.3 μm was obtained.

[0143] The obtained 2.3 - micron particles were then coated with glycidoxypropyltrimethoxysilane (GPTMS). A 20 mM sodium acetate buffer (pH 5.5) was added to a round - bottom flask equipped with a thermometer, condenser, and mechanical stirrer at approximately 5 mL / gram of particles). After the solution was heated to 70 °C, GPTMS was added at 10.22 μmol / m 2The solution was added to the flask at the specified concentration and mixed at the specified temperature. After 1 hour, the particles were added to the flask (1 g particles / 5 mL buffer solution). The mixture was stirred at 70°C for 20 hours, after which the reaction was cooled to below 40°C and filtered. The product was washed three times with water and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirrer. Acetic acid (0.1 M) was added to the flask (5 mL / gram particles), and the slurry was heated at 70°C for 20 hours. The flask was cooled to below 40°C and filtered. The product was washed with water until the pH of the supernatant rose above 5, and then washed three times with methanol. The isolated particles were dried in a vacuum oven at 70°C for 16 hours.

[0144] Five grams of the obtained particles were added to a 1 L three-necked round-bottom flask equipped with an overhead stirring motor, stirring shift, stirring blades, a water-cooled condenser, a nitrogen inlet, and a probe-controlled heating mantle. 50 mL of anhydrous diglycylm (diethylene glycol dimethyl ether) was added. The flask was sealed and purged with nitrogen for 15 minutes with gentle stirring. 0.224 g of potassium tert-butoxide was added, and the reaction was raised to 70°C. To create a hydrophilic layer, a mixture of 0.75 g glycidol, 0.19 g glycerol triglycidyl ether, and 1.1 g anhydrous diglycylm was prepared separately and added to the particle mixture in four equal portions with 30-minute intervals. The reaction was maintained at 70°C for 20 hours, cooled to RT, and filtered. The obtained particles were washed six times consecutively with water and three times consecutively with methanol, and dried overnight at 45°C under vacuum. This procedure yielded a hydrophilic layer of 2–4% by weight of the total particles.

[0145] 5 g of the resulting particles having a hydrophilic coating were added RT-to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol. 0.25 mL of 50% sodium hydroxide in water was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed 10 times with methanol, and partially dried under a nitrogen stream. The particles were stored at 4°C on a methanol-moistened bed. The resulting particles have a sufficient epoxide content to enable functionalization of the particle surface.

[0146] The particles were functionalized with protein A as described in Example 2, yielding approximately 2.5 ug of protein A per mg of particle.

[0147] The ability of particles to bind to and elute monoclonal antibodies (NISTmAb standards, available from Sigma-Aldrich) was tested. The particles were packed into a 2.1 × 200 mm column and connected to an ultra-high-performance liquid chromatography system (Acquity Premier, Waters Technologies Corporation, available from Milford MA). The column was rapidly flushed with water (pH 4) at 0.5 mL / min for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. 10 μL injection solutions of monoclonal antibody standards at various concentrations (ranging from 0.001 to 1 μg / uL) were injected into the column at a flow rate of 1 mL / min using 0.1 M sodium phosphate (pH 7.4) buffer. Blank injection solutions of binding buffer were used between samples to calculate carryover. Elution was performed for 1 minute using either 24 mM phosphate (pH 2) or 100 mM glycine (pH 2.4). The column was equilibrated for 1 minute using 0.1 M sodium phosphate buffer (pH 7.4).

[0148] The detection limits were 0.01 ug for the phosphate elution method and 0.25 ug for the glycine elution method. The maximum applicable pressure was 3100 psi. Figure 11A shows the elution profile for 24 mM phosphate (pH 2), and Figure 11B shows the elution profile for 100 mM glycine (pH 2.4). Figure 11C shows the linear correlation between monoclonal antibody concentration and mean peak area for both elution methods. Carryover was less than 1% for glycine and 2–10% for phosphate. The hydrophilic layer exhibited some tailing, indicating nonspecific binding of the monoclonal antibody to the particle surface.

[0149] Non-porous polymer particles with diol coating (particle type F) Polymer-based nonporous particles were prepared. 65.1 grams of polyvinylpyrrolidone (PVP-40), 8.9 grams of Triton N-57, 2452.6 grams of reagent alcohol (90% ethanol, 5% methanol, 5% isopropanol), and 153.3 grams of p-xylene were charged into a 4 L cylinder flask reactor equipped with mechanical stirring, a condenser, and a thermocouple. The reaction was carried out with stirring at 200 rpm, and dissolved oxygen in the solution was removed by subsurface purging with nitrogen. After the dissolved oxygen level was reduced to less than 1 ppm, 2.1 grams of azobisisobutyronitrile and 105 grams of divinylbenzene were added to the reaction. The reaction was raised to 70°C and maintained for 20 hours.

[0150] 1.8 grams of azobisisobutyronitrile was added to the reaction, followed by the addition of a solution of 67.6 grams of divinylbenzene (DVB) and 20.3 grams of PVP-40 in 202.7 grams of reagent alcohol, which was added via a pump at a constant flow rate over 120 minutes. The reaction was continued at 70°C for at least 12 hours. The particles were separated from the reaction slurry by filtration and washed sequentially with methanol, tetrahydrofuran (THF), and acetone. The final product was dried overnight in a vacuum oven at 45°C.

[0151] 46.2 grams of polymer core were dispersed in 231 grams of ethanol by sonication for 4 minutes using an ultrasonic horn and transferred to a round-bottom flask equipped with a mechanical-grade stirrer, condenser, and thermocouple. 49.1 grams of ammonium hydroxide and 462 grams of toluene were added to the reaction and mixed at RT for 60 minutes. The reaction was raised to 35°C, and a mixture of 26.06 grams of TEOS and 66.34 grams of BTEE, diluted with 148.9 grams of ethanol and 148.9 grams of toluene, was charged at a constant flow rate of 0.95 mL / min via a peristaltic pump. After the reaction, the particles were separated by filtration, washed six times with methanol, and dried overnight in a vacuum oven at 45°C.

[0152] The obtained polymer core particles were dispersed in an aqueous solution of 0.3 M tris(hydroxymethyl)aminomethane at a slurry concentration of 5 mL / g. The pH was adjusted to 9.8 using acetic acid. The slurry was then sealed in a stainless steel autoclave and heated at 155°C for 20 hours. After cooling to RT, the product was isolated on 0.5 micron filter paper and washed with water and methanol. The particles were dried under vacuum at 80°C for 16 hours.

[0153] After the hot water step, 50 g of particles were refluxed for 10 hours in 1 M hydrochloric acid at a slurry concentration of 10 mL / g. The reaction was cooled to RT, and the particles were isolated by filtration. The particles were washed with water until the pH of the filtrate was above 5. The partially neutralized particles were washed three times with acetone (10 mL / g). The resulting particles were sized to remove aggregates, and a final particle size of 2.3 μm was obtained.

[0154] The resulting 2.3 micron particles were then coated with glycidoxypropyltrimethoxysilane (GPTMS). Approximately 5 mL / gram of 20 mM sodium acetate buffer (pH 5.5) was added to a round-bottom flask equipped with a thermometer, condenser, and mechanical stirrer. After the solution was heated to 70°C, the GPTMS was mixed to a concentration of 10.22 μmol / m³. 2The solution was added to the flask at the specified concentration and mixed at the specified temperature. After 1 hour, the particles were added to the flask (1 g particles / 5 mL buffer solution). The mixture was stirred at 70°C for 20 hours, after which the reaction was cooled to below 40°C and filtered. The product was washed three times with water and transferred to a new flask equipped with a thermometer, condenser, and mechanical stirrer. Acetic acid (0.1 M) was added to the flask (5 mL / gram particles), and the slurry was heated at 70°C for 20 hours. The flask was cooled to below 40°C and filtered. The product was washed with water until the pH of the supernatant rose above 5, and then washed three times with methanol. The isolated particles were dried in a vacuum oven at 70°C for 16 hours.

[0155] 5 g of the resulting particles having a hydrophilic coating were added RT-to a mixture of 25 g of ethylene glycol diglycidyl ether (EGDGE) and 25 g of methanol. 0.25 mL of 50% sodium hydroxide in water was added, and the reaction was continuously stirred for 20 hours. The particles were isolated by filtration, washed 10 times with methanol, and partially dried under a nitrogen stream. The particles were stored at 4°C on a methanol-moistened bed. The resulting particles have a sufficient epoxide content to enable functionalization of the particle surface.

[0156] The particles were functionalized with protein A as described in Example 2, yielding approximately 4.66 ug of protein A per mg of particle.

[0157] The ability of particles to bind and elute monoclonal antibodies (NISTmAb standards, available from Sigma-Aldrich) was tested. The particles were packed into a 2.1 × 200 mm column and connected to an ultra-high-performance liquid chromatography system (Acquity Premier, Waters Technologies Corporation, available from Milford MA). The column was rapidly flushed with water (pH 4) at 0.5 mL / min for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. 10 μL injection solutions of various concentrations of monoclonal antibody standards (ranging from 0.001 to 1 μg / uL) were injected into the column at a flow rate of 1 mL / min using 0.1 M sodium phosphate (pH 7.4) buffer. Blank injection solutions of binding buffer were used between samples to calculate carryover. Elution was performed for 1 minute with either 24 mM phosphate (pH 2) or 100 mM glycine (pH 2.4). The column was equilibrated for 1 minute using 0.1 M sodium phosphate buffer (pH 7.4).

[0158] The detection limit was 0.01 ug for the phosphate elution method and 0.25 ug for the glycine elution method. Retention times shifted to lower concentrations with glycine elution (see Figure 12B), which was not observed with the phosphate elution method (see Figure 12A). The maximum applicable pressure was 4900 psi. Figure 12C shows the linear correlation between monoclonal antibody concentration and mean peak area for both elution methods. Carryover was less than 1.5% for glycine and less than 6% for phosphate. Although low carryover was observed, considerable tailing was observed due to nonspecific binding of the monoclonal antibody to the particle surface.

[0159] Particles of Example 1 functionalized with Protein A (Particle Type A) For comparison, particles from Examples 1 and 2 were tested using the same binding and elution methods. The particles were packed into a 2.1 × 200 mm column and connected to an ultra-high-performance liquid chromatography system (Acquity Premier, Waters Technologies Corporation, available from Milford MA). The column was rapidly flushed with water (pH 4) at 0.5 mL / min for 10 minutes to remove unbound protein A. The column was then equilibrated in 0.1 M sodium phosphate (pH 7.5) at 0.5 mL / min for 10 minutes. 10 μL injection solutions of monoclonal antibody standards at various concentrations (ranging from 0.001 to 1 μg / uL) were injected into the column at a flow rate of 1 mL / min using 0.1 M sodium phosphate (pH 7.4) buffer. Blank injection solutions of binding buffer were used between samples to calculate carryover. Elution was performed for 1 minute with either 24 mM phosphate (pH 2) or 100 mM glycine (pH 2.4). The column was equilibrated for 1 minute using 0.1 M sodium phosphate buffer (pH 7.4).

[0160] The detection limits were 0.01 ug for the phosphate elution method and 0.1 ug for the glycine elution method. The maximum applicable pressure was 5000 psi. Carryover was less than 2% for glycine and less than 6% for phosphate. High sensitivity and detector saturation (10 ug) were observed with low amounts of monoclonal antibody, as shown in Figure 13A (24 mM phosphate (pH 2)) and Figure 13B (100 mM glycine (pH 2.4)). Figure 13C shows the linear correlation between monoclonal antibody concentration and mean peak area.

[0161] Comparison of particle types A to H Figures 14A–14B provide a comparison of peak shapes in the highest concentration tested sample (5 ug of monoclonal antibody) using the 24 mM phosphate elution method (Figure 14A) and the 100 mM glycine elution method (Figure 14B). As shown in Figure 14A, in the phosphate elution method, particle types B, C, D, and E showed retention shifts along with peak tailing, while particle type H exhibited peak splitting. Similarly, as shown in Figure 14B, particle types B, C, D, E, and F showed retention shifts. Particle types B, H, C, D, and E also had broadened peaks along with peak tailing. In particular, particle type A produced a narrow peak with higher intensity compared to the other materials tested.

[0162] Figure 15A shows the peak heights for particle types A, B, C, D, E, F, and G (porous particles described in Example 4). Figure 15B shows the peak volumes for the aforementioned particles. As shown in Figure 15A, particle type A has a peak height of >1.4X compared to all other particle types tested for both elution methods. Particle types A, C, D, E, and F have similarly low peak volumes. Without being constrained by theory, the low peak volumes may be partly attributable to small non-porous particles with increased dynamics. In contrast, particle type G had a remarkably high peak volume, i.e., a peak volume almost 4X higher than particle type A.

Claims

1. A chromatography column comprising a column body formed of metal or a metal alloy, The column body contains multiple particles, and each of the multiple particles is Non-porous polymer core, Hydrophilic surface on the outer layer of a non-porous polymer core, and One or more molecules of immunoglobulin-binding proteins conjugated on a hydrophilic surface Includes, A chromatography column in which particles have an average particle size of 1.5 μm to 8 μm.

2. The particle according to claim 1, wherein the immunoglobulin-binding protein is selected from the group consisting of protein A, protein G, protein A / G, protein L, or their binding domains.

3. The particle according to claim 1 or 2, wherein the non-porous polymer core has a gradient composition.

4. The particle according to any one of claims 1 to 3, wherein the non-porous polymer core contains divinylbenzene (80%).

5. The particle according to any one of claims 1 to 4, wherein the hydrophilic surface is selected from the group consisting of (3-glycidyloxypropyl)trimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, polyacrylate, glycidol, glycerol triglycidyl ether, butyl diglycidol ether, and poly(methyl acrylate).

6. The particle according to any one of claims 1 to 5, wherein one or more molecules of immunoglobulin-binding proteins are conjugated to the hydrophilic surface of the particle via an epoxy linker.

7. The particle according to any one of claims 1 to 6, wherein the average particle size is 2 μm to 5 μm.

8. The particle according to claim 7, wherein the average particle size is 3.5 μm.

9. The particle according to any one of claims 2 to 8, wherein the immunoglobulin-binding protein has a surface coating concentration of 3 to 9 μg of immunoglobulin-binding protein per mg of particle.

10. The particle according to claim 9, wherein the immunoglobulin-binding protein is protein A.

11. A chromatography column according to any one of claims 1 to 10, wherein at least a portion of the inner surface of the column body is coated with an alkylsilyl material.

12. The chromatography column according to claim 11, further comprising frit within the column body, and coated with an alkylsilyl material.

13. The chromatography column according to claim 11 or 12, wherein the alkylsilyl material is a hydrophilic, nonionic layer of polyethylene glycol silane.

14. A chromatography column according to any one of claims 1 to 13, A column injector located upstream of the chromatography column, and piping located downstream of the chromatography column, with fluid connection between them. A chromatography device including, A chromatography device in which a portion of the inner surface of the column injector and a portion of the inner surface of the piping are coated with an alkylsilyl material.

15. A method for enriching immunoglobulins, a) Step of selecting a chromatography column, b) Wash the chromatography column with binding buffer, c) The step of applying a solution containing immunoglobulin to a chromatography column, and d) Wash the chromatography column with elution buffer so that immunoglobulins are eluted from the chromatography column. Includes, A chromatography column includes a column body made of metal or a metal alloy, the column body contains multiple particles, and each of the multiple particles is Non-porous polymer core, Hydrophilic surface on the outer layer of a non-porous polymer core, and One or more molecules of immunoglobulin-binding proteins conjugated on a hydrophilic surface Includes, A method in which the particles have an average particle size of 1.5 μm to 8 μm.

16. The method according to claim 15, wherein the immunoglobulin-binding protein is selected from the group consisting of protein A, protein G, protein A / G, and protein L, or their binding domains.

17. The method according to claim 15 or 16, wherein the immunoglobulin-binding protein has a surface coating concentration of 3 to 9 μg of immunoglobulin-binding protein per mg of particle.

18. The method according to claim 17, wherein the immunoglobulin-binding protein is protein A.

19. The method according to any one of claims 15 to 18, wherein the chromatography column is connected to a high-performance liquid chromatography (HPLC) system, an ultra-high-performance liquid chromatography (UHPLC) system, or a high-performance protein liquid chromatography (FPLC) system.

20. The method according to any one of claims 15 to 19, wherein the elution buffer is at least three orders of magnitude more acidic than the binding buffer.

21. The method according to any one of claims 15 to 20, wherein the binding buffer has a pH of 7.0 to 8.

0.

22. The method according to any one of claims 15 to 21, wherein the elution buffer has a pH of 1.3 to 3.

5.

23. The method according to claim 15, further comprising step e) detecting immunoglobulins with an ultraviolet spectrometer, a fluorescence spectrometer, and / or a mass spectrometry detector.

24. The method according to claim 23, further comprising step f) washing the chromatography column with a binding buffer.

25. The method according to claim 24, further comprising the step of repeating steps b) to e).

26. The method according to claim 15, further comprising step e) washing the chromatography column with a binding buffer.

27. The method according to claim 26, further comprising the step of repeating steps b) to d).

28. The method according to claim 24 or 26, wherein the chromatography column is washed with binding buffer for 10 minutes or less.

29. The method according to any one of claims 15 to 28, wherein the step of applying a solution containing immunoglobulin to a chromatography column (step c)) is performed for about 1 to 3 minutes.

30. The method according to claim 29, wherein the step of applying a solution containing immunoglobulin to a chromatography column is performed for about 1 minute.

31. The method according to claim 29 or 30, wherein the immunoglobulin is eluted from the column in a peak volume of less than 50 μL.

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